Resonator Networks for Improved Label Detection, Computation, Analyte Sensing, and Tunable Random Number Generation

ABSTRACT

The present disclosure provides resonator networks adapted to a variety of applications. The networks include fluorophores, quantum dots, dyes, plasmonic nanorods, or other optical resonators maintained in position relative to each other by a backbone (e.g., a backbone composed of DNA). The networks may exhibit optical absorption and re-emission according to specified temporal decay profiles, e.g., to provide temporally-multiplexed labels for imaging or flow cytometry. The networks can include resonators that exhibit a dark state, such that the behavior of the network can be modified by inducing the dark state in one or more resonators. Such networks could be configured as logic gates or other logical elements, e.g., to provide multiplexed detection of analytes by a single network, to permit the temporal decay profile of the network to be adjusted (e.g., to use the networks as a controllable random number generator), or to provide other benefits.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/521,192, filed Jun. 16, 2017. U.S. Provisional Patent ApplicationNo. 62/527,451, filed Jun. 30, 2017, and U.S. Provisional PatentApplication No. 62/551,616, filed Aug. 29, 2017 which are incorporatedherein by reference.

BACKGROUND

A variety of fluorophores, quantum dots, Raman dyes, and other opticallyactive substances can be incorporated into labels. Such labels can beused to determine the presence, location, amount, or other properties ofthe label and/or of an analyte to which the label is configured to bindin a sample. This can include illuminating the sample at one or moreoptical wavelengths and detecting light responsively reflected by,absorbed and fluorescently re-emitted by, or otherwise emitted from thelabel. A timing, a spectral content, an intensity, a degree ofpolarization, or some other property of light detected from the samplein response to illumination of the sample could be used to detect theidentity of the label in the sample. For example, a library of labels,differing with respect to an excitation spectrum, an emission spectrum,a susceptibility to photobleaching, or some other optical property,could be applied to the sample m order to detect the presence, location,or other properties of a respective plurality of analytes in the sample.

In some examples, a label can include multiple fluorophores insufficient proximity that energy can pass from an absorbing donorfluorophore of the label to an emitting acceptor fluorophore of thelabel. In such examples, a state of binding to a target analyte or someother status of such a label could be related to a distance between thedonor and acceptor. That is, the label binding to an instance of ananalyte could cause a conformation change in the label such that thedistance between the donor and acceptor increases (or decreases) to sucha degree that energy is less (or more) likely to transfer from the donorto the acceptor. In such examples, a degree of overall fluorescence ofthe label, or some other optical property of the label, could bedetected and used to determine the presence, location, amount, anisoform, or some other property of the analyte in a sample.

SUMMARY

One aspect of the present disclosure provides a label including: (i) twoor more input resonators that each include at least one of afluorophore, a quantum dot, or a dye; (ii) an output resonator thatincludes at least one of a fluorophore or a quantum dot; and (iii) anorganic backbone. The two or more input resonators and the outputresonator are coupled to the backbone and the backbone maintainsrelative locations of the input resonators and the output resonator suchthat energy can be transmitted from each of the input resonators to theoutput resonator.

Another aspect of the present disclosure provides a label including: (i)an input resonator; (ii) one or more mediating resonators, where a firstone of the one or more mediating resonators is disposed proximate to theinput resonator such that the first mediating resonator can receiveenergy from the input resonator; (iii) an output resonator, where atleast one of the one or more mediating resonators is disposed within thelabel proximate to the output resonator such that the output resonatorcan receive energy from the at least one of the one or more mediatingresonators; and (iv) a backbone. The input resonator, the outputresonator, and the one or more mediating resonators are coupled to thebackbone and the backbone maintains relative locations of the inputresonator, the output resonator, and the one or more mediatingresonators such that energy can be transmitted from the input resonatorto the output resonator via the one or more mediating resonators.

Another aspect of the present disclosure provides a system including;(i) a sample container; (ii) a light source; (iii) a light detector; and(iv) a controller. The controller is programmed to perform operationsincluding: (a) illuminating, using the light source, the samplecontainer; (b) using the light detector, detecting a timing, relative tothe illumination of the sample container, of emission of a plurality ofphotons from the sample container within a range of detectionwavelengths, and (c) determining, based on the detected timing ofemission of the plurality of photons, an identity of a label.Determining the identity of the label includes selecting the identity ofthe label from a set of known labels. The label includes: (1) an inputresonator; (2) an output resonator, where the output resonator ischaracterized by an emission wavelength and the range of detectionwavelengths includes the emission wavelength of the output resonator;and (3) a network of one or more mediating resonators, where relativelocations of the input resonator, the output resonator, and the one ormore mediating resonators within the label are such that energy can betransmitted from the input resonator to the output resonator via the oneor more mediating resonators in response to the input resonator beingexcited by the illumination.

Yet another aspect of the present disclosure provides a non-transitorycomputer-readable medium having stored thereon instructions executableby at least one processor to perform functions including: (i)illuminating a sample that contains a label; (ii) detecting a timing,relative to the illumination of the sample, of emission of a pluralityof photons from the sample within a range of detection wavelengths,where the range of detection wavelengths includes an emission wavelengthof an output resonator of the label; and (iii) determining, based on thedetected timing of emission of the plurality of photons, an identity ofthe label. The label includes: (a) an input resonator; (b) an outputresonator, where the output resonator is characterized by an emissionwavelength; and (c) a network of one or more mediating resonators, whererelative locations of the input resonator, the output resonator, and theone or more mediating resonators within the label are such that energycan be transmitted from the input resonator to the output resonator viathe network of one or more mediating resonators in response to the inputresonator being excited by the illumination. Determining the identity ofthe label includes selecting the identity of the label from a set ofknown labels.

Yet another aspect of the present disclosure provides a contrast agentincluding: (i) a first label; and (ii) a second label. The first labelincludes: (a) a first receptor that selectively interacts with a firstanalyte of interest; (b) at least two first input resonators; (c) atleast one first output resonator, where a ratio between a number offirst input resonators in the first label and a number of first outputresonators in the first label has a first value; and (d) a firstbackbone, where the first receptor, the at least two first inputresonators, and the at least one first output resonator are coupled tothe first backbone, and the first backbone maintains relative locationsof the at least two first input resonators and the at least one firstoutput resonator such that energy can be transmitted from each of thefirst input resonators to at least one first output resonator. Thesecond label includes: (a) a second receptor that selectively interactswith a second analyte of interest; (b) at least two second inputresonators; (c) at least one second output resonator, where a ratiobetween a number of second input resonators in the second label and anumber of second output resonators in the second label has a secondvalue; and (d) a second backbone, where the second receptor, the atleast two second input resonators, and the at least one second outputresonator are coupled to the second backbone, and the second backbonemaintains relative locations of the at least two second input resonatorsand the at least one second output resonator such that energy can betransmitted from each of the second input resonators to at least onesecond output resonator. Further, the first value and the second valuediffer

Yet another aspect of the present disclosure provides a methodincluding: (i) illuminating a sample that contains a label; (ii)detecting a timing, relative to the illumination of the sample, ofemission of a plurality of photons from the sample within a range ofdetection wavelengths, wherein the range of detection wavelengthsincludes an emission wavelength of an output resonator of the label; and(iii) determining, based on the detected timing of emission of theplurality of photons, an identity of the label. The label includes: (a)an input resonator; (b) an output resonator that is characterized by anemission wavelength; and (c) a network of one or more mediatingresonators, where relative locations of the input resonator, the outputresonator, and the one or more mediating resonators within the label aresuch that energy can be transmitted from the input resonator to theoutput resonator via the network of one or more mediating resonators inresponse to the input resonator being excited by the illumination.Determining the identity of the label includes selecting the identity ofthe label from a set of known labels.

Yet another aspect of the present disclosure provides a systemincluding: (i) a sample container, (ii) a light source; (iii) a lightdetector; and (iv) a controller. The controller is programmed to performoperations including: (a) illuminating, using the light source, thesample container; (b) using the light detector, detecting a timing,relative to the illumination of the sample container, of emission of aplurality of photons from the sample container within a range ofdetection wavelengths; and (c) determining, based on the detected timingof emission of the plurality of photons, an identity of a label.Determining the identity of the label includes selecting the identity ofthe label from a set of known labels. The label includes: an inputresonator that is characterized by an emission wavelength, where therange of detection wavelengths includes the emission wavelength of theinput resonator; and (b) a modulating resonator, where relativelocations of the input resonator and the modulating resonator within thelabel are such that energy can be transmitted between the inputresonator and the modulating resonator in response to the inputresonator being excited by the illumination.

Yet another aspect of the present disclosure provides a resonatornetwork including: (i) a first input resonator that has a dark state,where the first input resonator can enter the dark state in response toreceiving illumination at a first input excitation wavelength; (ii) areadout resonator that can receive energy from illumination at a readoutwavelength; (iii) an output resonator; and (iv) a backbone. The firstinput resonator, the readout resonator, and the output resonator arecoupled to the backbone, and the backbone maintains relative locationsof the first input resonator, the readout resonator, and the outputresonator such that energy can be transmitted from the readout resonatorto the output resonator and further such that a probability of energybeing transmitted from the readout resonator to the output resonator isrelated to whether the first input resonator is in the dark state.

Yet another aspect of the present disclosure provides a method fordetecting an analyte, the method including: (i) illuminating a resonatornetwork, during a first period of time, with light at a first inputwavelength; (ii) illuminating the resonator network, during the firstperiod of time, with light at a readout wavelength; and (iii) detecting,during the first period of time, an intensity of light emitted from anoutput resonator of the resonator network. The resonator networkincludes: (a) a first input resonator that has a dark state, where thefirst input resonator can enter the dark state in response to receivingillumination at the first input excitation wavelength; (b) a readoutresonator that can receive energy from illumination at the readoutwavelength; (c) a mediating resonator; (d) an output resonator; (e) asensor that includes a receptor that preferentially binds to an analyte;and (f) a backbone. The first input resonator, the readout resonator,the sensor, and the output resonator are coupled to the backbone, andthe backbone maintains relative locations of the first input resonator,the readout resonator, the mediating resonator, the sensor, and theoutput resonator such that energy can be transmitted from the readoutresonator to the output resonator via the mediating resonator andfurther such that a probability of energy being transmitted from thereadout resonator to the output resonator, when the first inputresonator is in the dark state, is related to whether the receptor isbound to an instance of the analyte.

Yet another aspect of the present disclosure provides a methodincluding: (i) illuminating a plurality of resonator networks, during afirst period of time, with light at a first input wavelength; (ii)illuminating the plurality of resonator networks, during the firstperiod of time, with light at a readout wavelength, and (iii) detectinga timing, relative to the illumination of the resonator networks, ofemission of a plurality of photons from output resonators of theplurality of resonator networks. Each resonator network of the pluralityof resonator networks includes: (a) a first input resonator that has adark state and that can enter the dark state in response to receivingillumination at the first input excitation wavelength; (b) a readoutresonator that can receive energy from illumination at the readoutwavelength; (c) two or more mediating resonators; (d) an outputresonator; and (e) a backbone. The first input resonator, the readoutresonator, the two or more mediating resonators, and the outputresonator are coupled to the backbone, and the backbone maintainsrelative locations of the first input resonator, the readout resonator,the two or more mediating resonators, and the output resonator such thatenergy can be transmitted from the readout resonator to the outputresonator via the mediating resonator and further such that theresonator network emits photons from the output resonator, in responseto the readout resonator receiving illumination at the readoutwavelength, according to a time-dependent probability density function.A detectable property of the time-dependent probability density functionis related to whether the first input resonator is in the dark state.

These as well as other aspects, advantages, and alternatives, willbecome apparent to those of ordinary skill in the art by reading thefollowing detailed description, with reference where appropriate to theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic of resonators in a label.

FIG. 1B shows a state transition diagram of the label illustratedschematically in FIG. 1A.

FIG. 2A shows the cumulative probability, over time, that a variety ofterminal states of a label have occurred.

FIG. 2B shows the probability that a label will emit a photon as afunction of time following excitation of the label.

FIG. 3A shows a schematic of resonators in a label.

FIG. 3B shows a schematic of resonators in a label.

FIG. 3C shows a schematic of resonators in a label.

FIG. 3D shows a schematic of resonators in a label.

FIG. 3E shows a schematic of resonators in a label.

FIG. 3F shows a schematic of resonators in a label.

FIG. 4A shows the probability that a variety of different labels willemit a photon as a function of time following excitation of the labels.

FIG. 4B shows the count of photon received from samples of two differentlabels as a function of time following excitation of the labels.

FIG. 5 shows a schematic of an example label.

FIG. 6A shows a schematic of resonators in a label.

FIG. 6B shows a schematic of resonators in a label.

FIG. 6C shows a schematic of resonators in a label.

FIG. 6D shows a schematic of resonators in a label.

FIG. 6E shows a schematic of resonators in a label.

FIG. 6F shows a schematic of resonators in a label.

FIG. 6G shows a schematic of resonators in a label.

FIG. 6H shows a schematic of resonators in a label.

FIG. 7A shows a schematic of resonators in a network.

FIG. 7B shows a schematic of resonators in a network.

FIG. 7C shows a schematic of resonators in a network.

FIG. 7D shows a schematic of resonators in a network.

FIG. 8A shows a schematic of resonators in a network.

FIG. 8B shows a schematic of resonators in a network.

FIG. 8C shows a schematic of resonators in a network.

FIG. 8D shows a schematic of resonators in a network.

FIG. 8E shows a schematic of resonators in a network.

FIG. 8F shows a schematic of resonators in a network.

FIG. 9A shows a schematic of resonators in a network.

FIG. 9B shows a schematic of resonators in a network.

FIG. 10A shows a schematic of resonators in a network.

FIG. 10B shows a schematic of resonators in a network.

FIG. 11 shows a flow chart of an example method.

FIG. 12 shows a flow chart of an example method.

FIG. 13 shows a flow chart of an example method.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying figures, which form a part hereof. In the figures, similarsymbols typically identify similar components, unless context dictatesotherwise. The illustrative embodiments described in the detaileddescription, figures, and claims are not meant to be limiting. Otherembodiments may be utilized, and other changes may be made, withoutdeparting from the scope of the subject matter presented herein. It willbe readily understood that the aspects of the present disclosure, asgenerally described herein, and illustrated in the figures, can bearranged, substituted, combined, separated, and designed in a widevariety of different configurations, all of which are explicitlycontemplated herein.

I. OVERVIEW

DNA self-assembly and other emerging nano-scale manufacturing techniquespermit the fabrication of many instances of a specified structure withprecision at the nano-scale. Such precision may permit fluorophores,quantum dots, dye molecules, plasmonic nanorods, or other opticalresonators to be positioned at precise locations and/or orientationsrelative to each other in order to create a variety of optical resonatornetworks. Such resonator networks may be specified to facilitate avariety of different applications. In some examples, the resonatornetworks could be designed such that they exhibit a pre-specifiedtemporal relationship between optical excitation (e.g., by a pulse ofillumination) and re-emission; this could enable temporally-multiplexedlabels and taggants that could be detected using a single excitationwavelength and a single detection wavelength. Additionally oralternatively, the probabilistic nature of the timing of opticalre-emission, relative to excitation, by these resonator networks couldbe leveraged to generate samples of a random variable. These resonatornetworks may include one or more “input resonators” that exhibit a darkstate; resonator networks including such input resonators may beconfigured to implement logic gates or other structures to control theflow of excitons or other energy through the resonator network. Suchstructures could then be used, e.g., to permit the detection of avariety of different analytes by a single resonator network, to controla distribution of a random variable generated using the resonatornetwork, to further multiplex a set of labels used to image a biologicalsample, or to facilitate some other application.

These resonator networks include networks of fluorophores, quantum dots,dyes, Raman dyes, conductive nanorods, chromophores, or other opticalresonator structures. The networks can additionally include antibodies,aptamers, strands of deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), or other receptors configured to permit selective binding toanalytes of interest (e.g., to a surface protein, molecular epitope,characteristic nucleotide sequence, or other characteristic feature ofan analyte of interest). The labels could be used to observe a sample,to identify contents of the sample (e.g., to identity cells, proteins,or other particles or substances within the sample), to sort suchcontents based on their identification (e.g., to sort cells within aflow cytometer according to identified cell type or other properties),or to facilitate some other applications.

In an example application, such resonator networks may be applied (e.g.,by coupling the resonator network to an antibody, aptamer, or otheranalyte-specific receptor) to detect the presence of, discriminatebetween, or otherwise observe a large number of different labels in abiological or material sample or other environment of interest. Suchlabels may permit detection of the presence, amount, or location of oneor more analytes of interest in a sample (e.g., in a channel of a flowcytometry apparatus). Having access to a large library ofdistinguishable labels can allow for the simultaneous detection of alarge number of different analytes. Additionally or alternatively,access to a large library of distinguishable labels can allow for moreaccurate detection of a particular analyte (e.g., a cell type orsub-type of interest) by using multiple labels to bind with the sameanalyte, e.g., to different epitopes, surface proteins, or otherfeatures of the analyte. Yet further, access to such a large library oflabels may permit selection of labels according to the probable densityor number of corresponding analytes of interest, e.g., to ensure thatthe effective brightness of different labels, corresponding to analyteshaving different concentrations in a sample, is approximately the samewhen optically interrogating such a sample.

Such labels may be distinguishable by virtue of differing with respectto an excitation spectrum, an emission spectrum, a fluorescencelifetime, a fluorescence intensity, a susceptibility to photobleaching,a fluorescence dependence on binding to an analyte or on some otherenvironmental factor, a polarization of re-emitted light, or some otheroptical properties. However, it can be difficult to produce a largelibrary of distinguishable labels when relying on differences withrespect to emission or excitation spectrum due to limitations on theavailable fluorophores or other optical distinguishable substances andlimitations on the wavelength transparency/compatibility of commonsample materials of interest.

The present disclosure provides methods for specifying, fabricating,detecting, and identifying optical labels that differ with respect totemporal decay profile and/or excitation and emission spectra.Additionally or alternatively, the provided labels may have enhancedbrightness relative to existing labels (e.g., fluorophore-based labels)and may have a configurable brightness to facilitate panel design or topermit the relative brightness of different labels to facilitate someother consideration. Such labels can differ with respect to thetime-dependent probability of re-emission of light by the labelsubsequent to excitation of the label (e.g., by an ultra-fast laserpulse). Additionally or alternatively, such labels can include networksof resonators to increase a difference between the excitation wavelengthof the labels and the emission wavelength of the labels (e.g., byinterposing a number of mediating resonators between an input resonatorand an output resonator to permit excitons to be transmitted betweeninput resonators and output resonators between which direct energytransfer is disfavored). Yet further, such labels may include logicgates or other optically-controllable structures to permit furthermultiplexing when detecting and identifying the labels.

Since such labels may differ with respect to temporal decay profile,they may be detected and identified in a sample by illuminating thesample with a single wavelength of illumination and/or by detectingresponsively emitted light from the sample within a narrow band ofwavelengths. Such a detection paradigm could simplify apparatuses usedto interrogate samples containing such labels and/or could facilitatehigh-label-count interrogation of sample media having strict opticalrequirements (e.g., that exhibit significant auto-fluorescence, that areparticular sensitive to photobleaching or other deleterious opticaleffects, that have narrow bands of transparency).

Each label (or other resonator network as described herein) includes atleast one input resonator that is capable of receiving optical energy toexcite the network (e.g., energy from an interrogating laser pulse) andat least one output resonator that is capable of emitting a photon inresponse to receiving, via the resonator network, energy (e.g., as anexciton transmitted via Förster resonance energy transfer (FRET) and/orsome other mechanism) from the input resonator. The relative locationsof the input resonator(s), output resonator(s), and one or moreadditional mediator resonators permit the transfer of excitons,electrical fields, surface plasmons, or other energy from resonator toresonator such that, when a particular resonator of the network isexcited (e.g., the input resonator), it has a chance to transfer thatexcitation energy to one or more other resonators (e.g., the outputresonator). The number and arrangement of resonators present in eachinstance of such a label (e.g., a number of input resonators of eachinstance of a label) may be specified to set a brightness of the label(e.g., to normalize the intensity of light emitted from a sample bydifferent labels that may have bound to analytes present in the sample).

The temporal decay profile of a particular label may thus be related tothe properties of the resonator network, e.g., to the identity andproperties (e.g., probability of nonradiative decay, probability ofresonance energy transfer to another resonator, or probability ofradiative emission) of the resonators and the relative locations andorientations of the resonators within the network. For example, a numberof mediating resonators could be arranged sequentially between an inputresonator and an output resonator to form a resonator wire. The temporaldecay profile of such a resonator network could be related to the lengthof the wire, e.g., longer wires could exhibit decay profiles that havewider peaks situated later in time. A library of distinguishable labelscould be created by varying the properties of the resonance network foreach of the labels such that the corresponding decay profiles of thelabels are distinguishable. Thus, the presence, identity, or otherproperties of such labels in a sample could be detected by illuminatingthe sample and detecting a timing, relative to the illumination, ofemission of photons from the sample.

Additionally or alternatively, the probabilistic nature of the timedifference between excitation and re-emission of light from suchresonator networks may be leveraged to generate samples of a randomvariable. The temporal decay profile of such a resonator network couldbe static (that is, set by the structure of the network and not easilymodified or controlled); in such examples, the timing of photonre-emission from such a network (or from a population of such networks)could be used to generate samples of a single random variable that isrelated to the static temporal decay profile of the network.Alternatively, such a network could include one or more input resonatorsthat exhibit a dark state (i.e., that may be disabled, with respect totheir ability to transmit and/or receive energy to/from other resonatorsin the network) when appropriately optically stimulated. Such inputresonators may be used to adjust the temporal decay profile of thenetwork over time, e.g., to permit use of the resonator network togenerate samples of a variety of different random variables that arerelated to respective different, optically-controllable temporal decayprofiles of the network.

Such dark state-exhibiting resonators may be incorporated into thenetwork such that their being in a dark state inhibits and/orfacilitates transmission of energy between different portions (e.g.,between an input and an output) of the network. For example, such aninput resonator could be situated between two other resonators suchthat, when the input resonator was in a dark state, energy transmissionbetween the two other resonators, via the input resonator, is impeded.In another example, an input resonator could be placed within a networksuch that, when the input resonator was not in a dark state, the inputresonator preferentially received energy from one or more otherresonators in the network. Thus, placing the input resonator into thedark state could act to prevent the input resonator from “sinking”energy from the network.

Such dark state-exhibiting resonators may thus be incorporated into aresonator network in order to provide logical functions within thenetwork. For example, such a resonator network may be configured toexecute a logical computation, with inputs being “programmed” into thenetwork by inducing relevant input resonators to enter their dark states(e.g., by illuminating them with illumination at an excitationwavelength of the input resonator(s)). The logical computation couldthen be “read out” by optically exciting an additional resonator of thenetwork (a “readout resonator”) and detecting photons responsivelyemitted from an output resonator of the network.

Such resonator networks may also be used for sensing properties of asample or another environment of interest. e.g., to detect a presence oramount of one or more analytes of interest in a biological sample. Oneor more resonators of the network could be intrinsically sensitive to avariable of interest (e.g., a resonator could be quenched whenenvironmental pH is within a particular range). Additionally oralternatively, the network may include a sensor configured to alter oneor more detectable properties (e.g., a probability of re-emission inresponse to excitation, a temporal decay profile of excitation andre-emission) of the resonator network. For example, the network mayinclude a receptor (e.g., an antibody, an aptamer, a strand ofcomplementary DNA or RNA) that quenches a resonator of the network whenbound to an analyte, that quenches a resonator of the network when notbound to the analyte, that modifies a relative location of one or moreresonators of the network when bound to the analyte, or that otherwisemodifies the configuration and/or behavior of the resonator networkdepending on whether it is bound to an instance of the analyte. Such aresonator network may include logical elements (e.g., one or moredark-state-exhibiting resonators) such that a number of differentanalytes may be detected using a single resonator network (e.g., bycontrolling the dark-state input resonators to “address” a particularone of a variety of different receptors of the network).

The resonator network of such a label could be created via a variety oftechniques. In some examples, DNA self-assembly could be used to ensurethat the relative locations of the resonators within a label correspondto locations specified according to a desired temporal decay profile.For example, each resonator of the network could be coupled to arespective specified DNA strand. Each DNA strand could include one ormore portions that complement portions one or more other DNA strandssuch that the DNA strands self-assemble into a nanostructure thatmaintains the resonators at the specified relative locations.

II. LABELS USING SPECIFIED RESONATOR NETWORKS FOR TEMPORAL MULTIPLEXING

Labels as described herein can be created that distinguishably differwith respect to their temporal decay profiles in response toillumination. This can be accomplished by specifying the identity,number, relative location and/or orientation, topology, or otherproperties of a network of resonators of the label. These properties ofthe resonator can be specified such that the resulting temporal decayprofile corresponds to a desired temporal decay profile. For example,the resonator network of a label could be specified such that thetemporal decay profile of the label includes one or more peaks havingrespective specified widths, normalized amplitudes, mean delay times, orother properties or features such that the temporal decay profile of thelabel is distinguishable from one or more other labels and/or frombackground materials present in a sample or environment of interest(e.g., fluorescent proteins of a cell or other biological sample).

Generally, the resonator network of such labels includes at least oneinput resonator, one or more mediating resonators, and at least oneoutput resonator. The resonators may be fluorophores, Raman dyes,quantum dots, dyes, pigments, conductive nanorods or othernanostructures, chromophores, or other substances that can receiveenergy from and/or transmit energy to one or more other resonators inthe network in the form of an exciton, an electrical fields, a surfaceplasmon, or some other form of energy that may be transferred, in aunitary manner, from one resonator to another.

The at least one input resonator of the network can receive energy intothe network as a result of the label being illuminated (e.g., by a laserpulse having a wavelength corresponding to an excitation wavelength ofthe input resonator). The at least one output resonator of the networkcan transmit energy from the network in the form of a photon whosetiming of emission, relative to illumination of the label, may bedetected and used, along with a plurality of additional photons detectedfrom a sample (e.g., from additional instances of the label in thesample, or from the particular instance of the label as a result ofrepeated illumination of the sample), to identify the label. The inputresonator, output resonator, and one or more mediating resonators arearranged to form the resonator network such that energy (e.g., excitons)received into the network via the input resonator(s) can be transmittedthrough the network to the output resonator(s).

Note that the labeling of any particular resonator in a network as“input,” “mediating,” or “output” is meant to be non-limiting. Aparticular resonator of a network could act as a mediating resonator forone or more other resonators and could also act as an input resonatorand/or as an output resonator for the network. Further, a label asdescribed herein could include only two resonators (e.g., an “input”resonator and a “modulating” resonator) and could be interrogated asdescribed herein by exposing the label to illumination that can exciteat least the input resonator and by detecting the timing, relative tothe illumination, of emission of a plurality of photons responsivelyemitted from at least one of the input resonator or the modulatingresonator. The input resonator (e.g., a fluorophore, a conductivenanorod or other nanoparticles, a quantum dot) could be disposed withinthe label such that energy (e.g., excitons, electrical fields) cantransfer from the input resonator to the modulating resonator (e.g., afluorophore, a conductive nanorod or other nanoparticles, a quantum dot,a non-fluorescent optically absorptive molecule or substance) and/orfrom the modulating resonator back to the input resonator.

The identity of such a two-resonator label, or of some other label asdescribed herein that can emit light from the same resonator by whichthe label can receive energy from illumination, could then be determinedbased on the detected relative timing of the emission of the pluralityof photons. For example, the label could be identified by comparing thedetected timing to a set of known temporal decay profiles, wherein thelabel corresponds to one of the temporal decay profiles in the set ofknown temporal decay profiles. In such an example, the temporal decayprofile of the label could be adjusted by specifying the identity of theresonators and by precisely controlling the relative locations and/ororientations of the resonators (e.g., using DNA self-assembly).

The particular configuration of the resonators and of the resonatornetwork as a whole result in the timing of emission of photons from theoutput resonator (or from the input resonator, a mediating resonator, amodulating resonator, or some other resonator of the label), relative toillumination of the label, exhibiting a characteristic temporal delayprofile. Thus, the timing of emission of a plurality of photons from asample relative to illumination of the sample (during one or moreillumination periods) could be detected and used to identify the labelin the sample, or to identify one or more additional labels in thesample, based on the characteristic temporal decay profile(s) of thelabel(s) in the sample.

FIG. 1A illustrates a schematic of resonators, and potential energytransfers to and from those resonators, of an example label 100 asdescribed herein. The example label 100 includes an input resonator 110a, a mediating resonator 110 b, and an output resonator 110 c. The inputresonator 110 a can be excited by receiving illumination 101 from theenvironment of the label 100. Once excited, the input resonator 110 acan radiatively emit a photon 140 a, nonradiatively decay 130 a suchthat the energy is lost (e.g., as heat) to the environment, or transferenergy 120 a to the mediating resonator 110 b (e.g., via the Försterresonance energy transfer process). In response to being excited, themediating resonator 110 b can radiatively emit a photon 140 b,nonradiatively decay 130 b, transfer energy 120 b to the outputresonator 110 c, or transfer energy 125 a to the input resonator 110 a.The output resonator 110 c, in response to being excited, canradiatively emit a photon 140 c, nonradiatively decay 130 c, or transferenergy 125 b to the mediating resonator 110 b.

By way of example, the relative probability of the different energytransitions/transfers are indicated in FIG. 1A by the relative lineweight of their representative arrows. Thus, for the example label 100,it is most likely that the input resonator 110 a transfers energy to themediating resonator 110 b, that the mediating resonator 110 b transfersenergy to the output resonator 110 c, and that the output resonator 110c radiatively emits a photon 140 c.

The time-dependence of each transition from a particular resonator canbe represented by a random variable having a particular distributionover time. For example, the mediating resonator 110 b transferringenergy (e.g., transferring an exciton) to the output resonator 110 ccould occur according to an exponentially distributed random variable inthe time domain. These random variables, along with the structure andother properties of the resonator network of the label 100, can be usedto model the behavior of the label 100, e.g., using a continuous timeMarkov chain. Such a model can then be used to predict the overalltemporal decay profile of the label 100 from excitation of the inputresonator 110 a by the illumination 101 to emission of a photon 140 c bythe output resonator 110 c.

FIG. 1B illustrates a state diagram that could be used to model thepotential states of the label 100, according to the excitation states ofthe resonators. This model assumes that only one of the resonators ofthe label 100 can be excited, as only a single unit of energy (e.g., asingle exciton) is received from the illumination 101 via the inputresonator 110 a. This unit of energy can then be transferred between theresonators and/or can exit the resonator network (e.g., via emission ofa photon or by non-radiative decay processes). The model includes statesfor excitation of the input resonator 110 a (“Input Excited”), themediating resonator 110 b (“Mediator Excited”), and the output resonator110 c (“Output Excited”). The model also includes absorbing states fornon-radiative decay from each of the resonators (“input Decayed,”“Mediator Decayed,” and “Output Decayed”) and radiative photon emissionfrom each of the resonators (“Input Emitted,” “Mediator Emitted,” and“Output Emitted”).

The transition probabilities for each transition are also indicated.These transition probabilities can be related to the identity of theresonators (e.g., to their intrinsic fluorescence lifetime, Försterradius), to their relative location, distance, and/or orientation (e.g.,distance relative to the Förster radius of a pair of the resonators), orto some other properties of the label 100. Thus, the relative locationsand identities of the resonators within the resonator network can bespecified to control the transition probabilities and topology of themodel, and thus to control the predicted temporal decay profile of thelabel 100.

In an example, a label includes an Alexa Fluor 448 dye as an inputresonator and an Alexa Fluor 594 dye as an output resonator, with theinput resonator and output resonator located proximate to each othersuch that the input resonator can transmit energy, as an exciton, to theoutput resonator in response to the input resonator being excited byillumination (e.g., an ultrashort laser pulse) FIG. 2A illustrates theprobability, over time, that the input resonator (“AF448 Fluorescence”)has emitted a photon, that the input resonator has decayed (“AF448Nonradiative Decay”), that the output resonator (“AF594 Fluorescence”)has emitted a photon, and that the output resonator has decayed (“AF594Nonradiative Decay”). FIG. 2A also illustrates the probability, overtime, that at least one of these processes has occurred (“Excitonrelaxation”).

From these probabilities, we can determine the temporal decay profile ofthe label. This is illustrated in FIG. 2B as “AF594 Fluorescence.” Thus,if a plurality of instances of the label was present in the sampleand/or if a sample containing a single instance of the label wasilluminated a plurality of times, the timing of emission of photons fromthe output resonator (e.g., at an emission wavelength of the Alexa Fluor594 dye) would exhibit a distribution over time, relative toillumination of the sample, corresponding to the illustrated temporaldecay profile. Conversely, the timing of emission of photons from theinput resonator (e.g., at an emission wavelength of the Alexa Fluor 488dye) would exhibit a distribution over time, relative to illumination ofthe sample, corresponding to the other temporal decay profileillustrated in FIG. 2B (“AF488 Fluorescence”).

The temporal decay profile of a particular label at a particularwavelength (e.g., the emission wavelength of an output resonator of thelabel) could be controlled by specifying the topology, structure,resonators types, or other properties of the resonator network of thelabel. Thus, a library of different, distinguishable labels could becreated by specifying their respective resonator networks such thattheir temporal decay profiles are distinguishable (e.g., by a particulardetection apparatus having a particular temporal resolution fordetection of photons from a sample containing such labels) from eachother and/or from background processes (e.g., fluorescence) in a sampleor other environment of interest. This could include specifying thetemporal decay profiles to maximize or increase a measure of statisticaldivergence (e.g., a Kullback-Leibler divergence, a Jensen-Shannondivergence, a Bregman divergence, or a Fisher information metric)between the temporal decay profiles. Additionally or alternatively, thetemporal decay profiles could be specified to differ with respect to thetiming, width, shape, number, or other properties of one or more peakspresent in the temporal decay profiles.

A resonator network could be determined to provide a desired temporaldecay profile using a variety of methods. For example, heuristic methodscould be used to vary a number of resonators in a resonator wire of thenetwork, a number a parallel resonator wires in a network between aninput and an output of the network, an identity of resonators (e.g.,relative to excitation and/or emission spectra of the resonators) of thenetwork, or other properties of the network in order to provide relatedchanges in a number, width, or delay of peaks in a temporal decayprofile, an average delay of the temporal decay profile, or otherproperties of the temporal decay profile. Additionally or alternatively,automated methods like genetic algorithms, machine learning, or othertechniques could be used to specify the configuration of one or moreresonator network such that their temporal decay profiles aredistinguishable or to provide some other benefit. The temporal decayprofile of such labels could then be verified experimentally, and theexperimentally determined temporal decay profiles could be used toidentify the labels present in a sample or other environment ofinterest.

FIG. 3A illustrates a schematic of resonators, and potential energytransfers to and from those resonators, of an example label 300 a asdescribed herein. The example label 300 a includes an input resonator(“IN”), two mediating resonators (“M1” and “M2”), and an outputresonator (“OUT”). The input resonator can be excited by receivingillumination from the environment of the label 300 a (e.g., illuminationat an excitation wavelength of the input resonator). The two mediatingresonators are arranged as a resonator wire between the input resonatorand the output resonator. That is, the two mediating resonators arearranged such that each resonator in the wire can receive energy fromand/or transmit energy to two neighboring resonators. The number ofresonators within such a resonator wire could be specified in order toadjust a temporal decay profile of the label 300 a. e.g., to adjust adelay or width of a peak in the decay profile, to increase an averagedecay of the decay profile, or to adjust some other property of thetemporal decay profile.

By way of example, the relative probability of the different energytransfers between the resonators are indicated in FIG. 3A by therelative line weight of their representative arrows. Thus, for theexample label 300 a, it is more likely that the input resonatortransfers energy to the first mediating resonator (M1) than vice versa.It is also more likely that the second mediating resonator (M2)transfers energy to output resonator than vice versa. It isapproximately equally likely that the first mediating resonatortransfers energy to the second mediating resonator as it is that thesecond mediating resonator transfers energy to the first mediatingresonator. Thus, energy generally travels unidirectionally from theinput resonator to the mediating resonators and from the mediatingresonators to the output resonators. Conversely, energy may travelbidirectionally between the mediating resonators before being emitted asa photon from the output resonator (or from one of the mediatingresonators) or lost via non-radiative processes.

The label 300 a of FIG. 3A illustrates a label incorporating atwo-element resonator wire in which energy may be transferredbidirectionally between adjacent resonators in the wire. Suchbidirectional energy transfer could be accomplished by selecting theresonators in the wire such that the emission spectrum of the firstmediating resonator significantly overlaps with the excitation spectrumof the second mediating resonator, and vice versa. This could beachieved by selecting the same fluorophore (e.g., Alexa Fluor 594) forboth of the mediating resonators in the wire.

Alternatively, one or more pairs of mediating resonators in a resonatornetwork (e.g., adjacent resonators in a resonator wire) could bespecified such that energy generally travels unidirectionally betweenpairs of such resonators. FIG. 3B illustrates a schematic of an examplelabel 300 b that includes such a resonator pair. The example label 300 bincludes an input resonator (“IN”), two mediating resonators (“M1” and“M2”), and an output resonator (“OUT”). The relative probability of thedifferent energy transfers between the resonators are indicated in FIG.3B by the relative line weight of their representative arrows. Thus, forthe example label 300 b, it is more likely that the input resonatortransfers energy to the first mediating resonator (M1) than vice versa.It is also more likely that the first mediating resonator (M1) transfersenergy to the second mediating resonator (M2) than vice versa and morelikely that the second mediating resonator (M2) transfers energy tooutput resonator than vice versa Thus, energy generally travelsunidirectionally from the input resonator through the mediatingresonators to the output resonator. The temporal decay profile of such alabel 300 b could exhibit a narrower and/or less-delayed peak and/orcould exhibit an overall reduced average delay relative to the temporaldecay profile of the first example label 300 a.

A label as described herein could include multiple resonator wires(e.g., multiple resonator wires of similar or different compositionconnected between common input and output resonators) having arbitrarylengths and/or compositions. For example, FIG. 3C illustrates aschematic of an example label 300 c that includes a resonator wire ofarbitrary length (i.e., that includes “n” resonators). The example label300 c includes an input resonator (“IN”), “n” mediating resonators(“M1,” “M2,” “M3,” “M4, . . . ” and “Mn”), and an output resonator(“OUT”). As indicated in FIG. 3C by the relative line weight of therepresentative arrows, energy transfers between adjacent mediatingresonators in the resonator wire are bidirectional. However, one or moreof the transitions between pairs of resonators of such a resonator wirecould be unidirectional.

The resonator network of a label as described herein could representdifferent topologies, e.g., a branched topology. Such a branchedtopology could include multiple different resonator wires whose ends areconnected to input resonators, output resonators, mediating resonators(e.g., an end resonator of one or more other resonator wires), orconnected in some other way to provide a label exhibiting a desiredtemporal decay profile.

FIG. 3D illustrates a schematic of an example label 300 d that includestwo paths by which energy can travel through the resonator network to beemitted by an output resonator. The example label 300 d includes aninput resonator (“IN”), a first mediating resonators (“M1”) that canreceive energy from the input resonator and that can transmit energy tothe output resonator, and three additional resonators (“M2,” “M3,” and“M4”) arranged as a resonator wire that can transmit energy from theinput resonator to the output resonator. As indicated by the relativeline weight of the representative arrows, energy transfers betweenadjacent mediating resonators in the resonator wire are bidirectional.Such a resonator network could exhibit a temporal decay profile that isa mixture of other temporal decay profiles, e.g., that is a mixture of afirst temporal decay profile of a label that only included the input,output, and first resonators and a second temporal decay profile of alabel that only included the input, output, and resonator wire (i.e.,mediating resonators “M2,” “M3,” and “M4”). A resonator network couldinclude a two- or three-dimensional field of mediating resonators, inputresonators, and/or output resonators. Such an arbitrary resonatornetwork could be determined via a genetic algorithm or other automatedprocess to provide a desired temporal decay profile or to satisfy someother criteria.

The resonator network of a label as described herein could includemultiple input resonators and/or multiple output resonators. Suchmultiple input and/or output resonators could be provided to provide avariety of benefits, e.g., to adjust an effective temporal decay profileof the label, to increase a probability that the label is excited inresponse to illumination and/or to increase the effective brightness ofthe label, to provide wavelength-dependent multiplexing to theexcitation and/or detection of the label (e.g., by causing the label toexhibit a different temporal decay profile, depending on which of anumber of spectrally-distinct input resonators is excited), or toprovide some other benefits. Multiple input resonators could be the same(that is, could each include the same fluorophores, quantum dots, orother optical elements) or could differ (e.g., could be differentfluorophores such that the different input fluorophores are excited byrespective different wavelengths of light). Multiple output resonatorscould be the same (that is, could each include the same fluorophores,quantum dots, or other optical elements) or could differ (e.g., could bedifferent fluorophores such that the different output fluorophores emitlight at respective different wavelengths). Additionally oralternatively, a single instance of a label could include multipledistinct or inter-connected resonator networks (e.g., multiple copies ofthe same resonator network) in order to increase and/or control theeffective brightness of the label, to reduce a time and/or number oflight pulses necessary to identify the label, or to provide some otherbenefit.

FIG. 3E illustrates a schematic of an example label 300 e that includesan input resonator (“IN”), two mediating resonators (“M1” and “M2”), anda first output resonator (“OUT1”). The label additional optionallyincludes second (“OUT2”) and third (“OUT3”) output resonators. Theadditional output resonators could be provided to adjust a temporaldecay profile of the label 300 e. For example, the second outputresonator could be the same as the first output resonator (e.g., couldhave the same emission spectrum) and could be added to the label 300 eto increase a probability that the label 300 e emits energy subsequentto the second mediating resonator being excited (e.g., by doubling theprobability that energy from the second mediating resonator istransferred to one of the first or second output resonators such thatone of the output resonators may then emit the received energy as aphoton).

Additionally or alternatively, additional output resonators could differwith respect to emission wavelength or emission spectrum and could beprovided to facilitate spectrally multiplexed detection of temporaldecay profiles at different wavelengths corresponding to the differentoutput resonators. For example, the third output resonator could differfrom the first output resonator (e.g., could have a different emissionspectrum) and could be added to the label 300 e such that the label 300e could emit a photon from one or the other of the output resonators.Such photons, differing with respect to wavelength, could be separatelydetected and used to determine two different temporal decay profiles forthe label 300 e (or from a sample containing the label) and suchmultiple detected temporal decay profiles could be used to identify thelabel 300 e.

FIG. 3F illustrates a schematic of an example label 300 f that includesa first input resonator (“IN1”), two mediating resonators (“M1” and“M2”), and an output resonator (“OUT”). The label additional optionallyincludes second (“IN2”) and third (“IN3”) input resonators. Theadditional input resonators could be provided to adjust a temporal decayprofile of the label 300 f or to increase the probability that the label300 f is excited by exposure to illumination. For example, the secondinput resonator could be the same as the first input resonator (e.g.,could have the same excitation spectrum) and could be added to the label300 f to increase a probability that the label 300 f receives energy inresponse to illumination (e.g., by doubling the probability that aphoton of the illumination is absorbed by at least one of the first orsecond input resonators).

Additionally or alternatively, additional input resonators could differwith respect to excitation wavelength or excitation spectrum and couldbe provided to facilitate spectrally multiplexed excitation of the label300 f and thus to facilitate spectrally multiplexed detection oftemporal decay profiles at different wavelengths corresponding to thedifferent input resonators. For example, the third input resonator coulddiffer from the first input resonator (e.g., could have a differentexcitation spectrum) and could be added to the label 300 f such that thelabel 300 f could be excited, during first and second periods of time,by first and second illumination which differ with respect to wavelengthand which are provided during the first and second periods of time,respectively. Such excitations of the label 300 f, differing withrespect to the input resonator by which the label 300 f was excited,could be characterized by respective different temporal decay profilesand thus detected, during separate periods of time, and used todetermine two different temporal decay profiles for the label 300 f (orfrom a sample containing the label) and such multiple detected temporaldecay profiles could be used to identify the label 300 f.

Note that the resonator networks of the labels described herein may alsobe employed to generate samples of a random variable. The sample of therandom variable may be generated based on a difference in time betweenexcitation of the resonator networks/labels and a timing of detection ofone or more photons responsively emitted from the resonatornetworks/labels. The particular distribution of the random variablecould be related to the temporal decay profile of the resonatornetworks/labels. For example, the value of the generated sample could bea function of a detected time difference between a timing ofillumination of the resonator network(s) and the timing of detection ofone or more photons responsively emitted from the resonator network(s).A distribution of the generated random variable could be related to thetemporal decay profile of the resonator network(s) and the functionapplied to generate samples of the random variable from the detectedtime difference. The structure of the resonator network(s) could bespecified (e.g., to exhibit a particular temporal decay profile or othertime-dependent probability density function) such that the function togenerate samples from detected time differences is computationallytractable and/or efficient to compute.

III. EXAMPLE SYSTEMS AND METHODS FOR IDENTIFYING LABELS IN A SAMPLE

It can be beneficial in a variety of applications to interrogate asample (e.g., a biological sample, or a stream of cells in a flowcytometer) or some other environment of interest in order to detect thepresence, identity, absolute or relative amount, or other properties oflabels as described herein that may be present in the sample or otherenvironment of interest. Such interrogation could facilitate imaging ofa sample. e.g., to determine the location, concentration, or otherinformation about one or more analytes that are present within thesample and to which one or more varieties of labels are configured tobind. Such interrogation could facilitate the identification of cells,proteins, strands of RNA, or other contents of a sample in order to sortsuch contents or to provide some other benefit. For example, a flowcytometry apparatus could include a flow channel through which cells (orother particles of interest) flow. Such a flow channel could beinterrogated as described herein in order to identify one or more labelsin the channel and/or to identify the type or subtype of the cells, todetermine a property of the cells, or to determine some otherinformation based on the identified labels. Such information could thenbe used to sort the cells. e.g., according to cell type.

Such methods for detecting and/or identifying labels in an environmentof interest could include providing illumination to the environment ofinterest (e.g., in the form of one or more ultrashort pulses ofillumination) and detecting one or more properties of photons emittedfrom the environment in response to the illumination (e.g., a wavelengthor spectrum of such photons, a timing of emission of such photonsrelative to a timing of the illumination. e.g., of one or more pulses ofthe illumination). This could include providing a single pulse ofillumination and detecting the photons responsively emitted from aplurality of instances of one or more labels in the environment.Additionally or alternatively, one or more instances of one or morelabels could be illumination a plurality of times by a plurality ofpulses of illumination and the timing, relative to the pulses ofillumination, of responsively emitted photons could be detected.Information about the timing of the responsively emitted photons couldthen be used to identify one or more labels that are present in theenvironment, to determine a binding state or other properties of suchlabels, to determine absolute or relative amounts of the label(s) in theenvironment, or to determine some other information related to one ormore labels as described herein that are present in the environment.

Illumination could be provided to an environment as one or more pulsesof illumination. The provided illumination could have a specifiedwavelength, e.g., an excitation wavelength of an input resonator of oneor more of the labels. Such an excitation wavelength could be commonacross some or all of the labels present in the environment of interest,e.g., due to some or all of the labels including the same fluorophore,quantum dot, dye, or other optical substance or structure as their inputresonator(s). Additionally or alternatively, multiple differentwavelengths of light could be provided to excite multiple differentinput resonators, e.g., of multiple different labels. In some examples,such different wavelengths of light could be provided at differentpoints in time (e.g., as part of different pulses of illumination) tofacilitate spectrally-multiplexed detection of multiple different labelsand/or multiple different sets of labels. In some examples, a singlelabel could include multiple different input resonators, and thedifferent input resonators could be excited by light at respectivedifferent wavelengths, e.g., as part of respective different pulses ofillumination.

In order to improve the identification of labels in an environment,pulses of illumination used to interrogate the environment could beultrashort pulses (e.g., pulses having durations on the order ofattoseconds to nanoseconds). Such ultrashort pulses could be provided asbroadband pulses emitted from a mode-locked oscillator. In exampleswherein a label includes resonators having long-lifetime states (e.g.,lanthanide atoms or other lanthanide compounds or complexes), the pulsesof illumination could have longer durations, e.g., on the order ofmicroseconds.

The timing, relative to such a pulse of illumination, of emission ofphotons from the environment in response to the pulse of illuminationcould be detected in a variety of ways. In some examples, the timing ofindividual photons could be detected, e.g., using one or moresingle-photon avalanche diodes, photomultipliers, or other single-photondetectors. The outputs of such detectors could be used, as part of atime-correlated single photon counter, to determine a count of photonsdetermined as a function of time after a pulse of illumination isprovided to the environment. The timing of such detected photons couldbe used to determine a probability density function for the timing ofemission of photons from the sample in response to illumination of thesample.

Additionally or alternatively, detecting the timing of emission ofphotons from the environment could include detecting a timing of one ormore peaks in the rate or intensity of the emitted photons, or detectingsome other aggregate property of the timing of the emitting photons(e.g., to determine a delay timing of a peak of the rate of emission ofphotons that could be matched to the delay of a corresponding peak of aknown temporal decay profile). Such detection could include applying apeak detector, a differentiator, a matched filter, or some other analogor digital signal processing techniques to the output of a single-photonavalanche diode or other photodetector element that is configured toreceive photons emitted from the environment of interest.

One or more known labels could be present in an environment of interestand it could be beneficial to determine the identity of such labelsand/or to determine some other information about the labels in theenvironment. As described above, such labels could be distinguishedaccording to their temporal decay profiles; that is, each known labelcould be characterized by a respective different temporal decay profile.Thus, the identity of the one or more labels present in the environmentcould be determined by comparing the detected timing of emission ofphotons from the environment to a dictionary of temporal decay profiles,where each of the temporal decay profiles in the dictionary correspondsto a respective known label that could be present in the environment.

FIG. 4A shows six different temporal decay profiles, each correspondingto one of six known labels. Each of the known labels has the same inputresonator (e.g., Alexa Fluor 430) and output resonator (e.g., AlexaFluor 750) which form a resonator wire in combination with one or moreof the same mediating resonator (e.g., Alexa Fluor 594). The knownlabels differ with respect to the number of mediating resonators.Information about the timing of photons received from an environmentcould be compared to the temporal decay profiles and used to determinewhich, if any, of the known labels are present in the environment. Thiscould include comparing a delay of a peak rate of emission of photonsfrom the environment to a delay of the peak in each of the knowntemporal decay profiles.

Additionally or alternatively, the detected timing of emission ofphotons could be used to determine a probability density function forthe timing of emission of photons from the sample in response toillumination of the sample. Such a determined probability densityfunction could then be compared to the temporal decay profiles of theknown labels and used to identify, one or more labels present in theenvironment. FIG. 4B illustrates the counts of photons detected from twodifferent samples over time in a number of discrete ranges of timerelative to illumination of the samples (at time 0). First counts(illustrated by the black rectangles) were received from a first samplethat contained known label “6” from FIG. 4A, and second counts(illustrated by the white rectangles) were received from a second samplethat contained known label “1” from FIG. 4A. The counts could be used todetermine respective first and second probability density functions forthe first and second samples, and the first and second probabilitydensity functions could be compared to the six known temporal decayprofiles in order to identify which of the known labels are present ineach of the samples. Such a comparison could include determining ameasure of statistical divergence (e.g., a Kullback-Leibler divergence,a Jensen-Shannon divergence, a Bregman divergence, or a Fisherinformation metric) between a determined probability density functionand each of the known temporal decay profiles. The label present in asample could then be determined, e.g., by selecting the known labelcorresponding to the least of the determined measures of statisticaldivergence.

Similar or different methods could be used to determine whether multiplelabels are present in a sample, and if so, to identify such multiplelabels. In some examples, the identity of a cell or other contents of anenvironment (e.g., of a flow channel of a flow cytometry apparatus)could then be determined based on the identity of the labels in theenvironment, e.g., based on the determination that all of a subset ofknown labels are simultaneously present in a flow channel or otherenvironment of interest.

In order to determine how many of a set of known labels are present inan environment, and to identify such present labels, a variety ofmethods can be used. For example, an expectation maximization algorithmcan be used, in concert with a statistical mixture model, to determinethe most likely labels present in an environment based on a determinedprobability density function for the timing of emission of photons fromthe environment in response to illumination of the environment. Such amixture model could be based on the set of temporal decay functionscorresponding to the set of known labels. Such an expectationmaximization algorithm and mixture model could also be used to determinethe relative amounts of such multiple labels in the sample.

Interrogating an environment could include detecting the timing ofemission of photons within multiple different ranges of wavelengths.This could be done to detect the timing of emission of photons from twodifferent output resonators of a label. Additionally or alternatively,this could be done to detect the timing of emission of photons from anoutput resonator, one or more mediating resonators, and/or an inputresonator of the label.

Yet further, one or more of the labels present in the environment mayinclude dark-state-exhibiting resonators such that the temporal decayprofile of the labels is dependent on whether the dark-state-exhibitingresonators are in their respective dark states. For example, a labelcould include a first dark-state-exhibiting resonator and could exhibita first temporal decay profile when the first dark-state-exhibitingresonator is in its dark state and the label could exhibit a secondtemporal decay profile when the first dark-state-exhibiting resonator isnot in its dark state. In such examples, detection and/or identificationof the label could include detecting a timing of optical excitation andre-emission during a time period when the dark-state-exhibitingresonator(s) is not in its dark state and, during a different period oftime, inducing the dark-state-exhibiting resonator(s) to enter the darkstate (e.g., by providing illumination at an excitation wavelength ofthe dark-state-exhibiting resonator(s)) and again detecting a timing ofoptical excitation and re-emission of the label.

IV. EXAMPLE RESONATOR NETWORKS

Resonator networks (e.g., resonator networks included as part of labels)as described herein can be fabricated in a variety of ways such that oneor more input and/o readout resonators, output resonators,dark-state-exhibiting “logical input” resonators, and/or mediatingresonators are arranged according to a specified network of resonatorsand further such that a temporal decay profile of the network, abrightness of the network, an excitation spectrum, an emission spectrum,a Stokes shift, or some other optical property of the network, or someother detectable property of interest of the network (e.g., a state ofbinding to an analyte of interest) corresponds to a specificationthereof (e.g., to a specified temporal decay profile, a probability ofemission in response to illumination). Such arrangement can includeensuring that a relative location, distance, orientation, or otherrelationship between the resonators (e.g., between pairs of theresonators) correspond to a specified location, distance, orientation,or other relationship between the resonators.

This can include using DNA self-assembly to fabricate a plurality ofinstances of one or more resonator networks. For example, a number ofdifferent DNA strands could be coupled (e.g., via a primary aminomodifier group on thymidine to attach an N-Hydroxysuccinimide (NHS)ester-modified dye molecule) to respective resonators of a resonatornetworks (e.g., input resonators, output resonator, and/or mediatorresonators). Pairs of the DNA strands could have portions that are atleast partially complementary such that, when the DNA strands are mixedand exposed to specified conditions (e.g., a specified pH, or aspecified temperature profile), the complementary portions of the DNAstrands align and bind together to form a semi-rigid nanostructure thatmaintains the relative locations and/or orientations of the resonatorsof the resonator networks.

FIG. 5 shows a schematic of such a resonator networks. An inputresonator (“SOURCE ATTO 488”), an output resonator (“EMITTER ATTO 610)and two mediator resonators (“MEDIATOR 1 ATTO 565” and “MEDIATOR 2 ATTO565”) are coupled to respective DNA strands. The coupled DNA strands,along with additional DNA strands, then self-assemble into theillustrated nanostructure such that the input resonator, mediatorresonators, and output resonator form a resonator wire. In someexamples, a plurality of separate identical or different networks couldbe formed, via such methods or other techniques, as part of a singleinstance of a resonator network (e.g., to increase a brightness of theresonator network).

The distance between resonators of such a resonator network could bespecified such that the resonator network exhibits one or more desiredbehaviors (e.g., is excited by light at a particular excitationwavelength and responsively re-emits light at an emission wavelengthaccording to a specified temporal decay profile) This can includespecifying the distances between neighboring resonators such that theyare able to transmit energy between each other (e.g., bidirectionally orunidirectionally) and further such that the resonators do not quencheach other or otherwise interfere with the optical properties of eachother. In examples wherein the resonators are bound to a backbone vialinkers (e.g., to a DNA backbone via an amide bond (created, e.g., byN-Hydroxysuccinimide (NHS) ester molecules) or other linkingstructures), the linkers could be coupled to locations on the backgroundthat are specified with these considerations, as well as the length(s)of the linkers, in mind. For example, the coupling locations could beseparated by a distance that is more than twice the linker length (e.g.,to prevent the resonators from coming into contact with each other, andthus quenching each other or otherwise interfering with the opticalproperties of each other). Additionally or alternatively, the couplinglocations could be separated by a distance that is less than a maximumdistance over which the resonators may transmit energy between eachother. For example, the resonators could be fluorophores or some otheroptical resonator that is characterized by a Förster radius whentransmitting energy via Förster resonance energy transfer, and thecoupling locations could be separated by a distance that is less thanthe Förster radius.

V. LABELS USING SPECIFIED RESONATOR NETWORKS FOR IMPROVED BRIGHTNESSAND/OR SPECTRAL MULTIPLEXING

When designing or specifying a set of resonator networks and/or labelsfor flow cytometry, molecular imaging, optical computation, biosensing,analyte assays, optical random number generation, or some otherapplication (e.g., via a process of panel design), it can be beneficialto be able to arbitrarily select the excitation spectrum/wavelength,emission spectrum/wavelength, extinction coefficient, brightness, orother optical properties of one or more resonator networks. Acombination of such resonator-network-containing labels (e.g., acontrast agent that includes two or more such labels) could then beapplied to a sample in order to detect, identify, image, or otherwiseobserve respective analytes of interest in a sample (e.g., by mixing orotherwise applying the multi-label contrast agent to the sample). Theability to detect, distinguish, or otherwise observe such labels in asample could be improved by selecting respective excitation wavelengths,emission wavelengths, brightnesses, extinction coefficients, absorptioncross-sections, or other optical properties of the different labelsapplied to the sample. Such labels can be created, as described herein,to differ with respect to their excitation spectrum, their emissionspectrum, their brightness, or other optical properties. This can beaccomplished by specifying the identity, number, relative locationand/or orientation, topology, or other properties of a network ofresonators of the label.

For example, it can be beneficial to select and/or configure differentlabels to differ with respect to excitation wavelength, emissionwavelength, Stokes shift or other spectral properties in order tofacilitate identification of such labels. Such identification could bebased on a detected wavelength of light emitted therefrom and/or on adetected or determined brightness of light emission from the label as afunction of the wavelength of light used to excite the label. However,when using single-resonator labels (e.g., single-fluorophore labels),the selection of such optical properties may be constrained by a limitedlibrary of commercially or otherwise available resonators. Usingtwo-resonator labels (e.g., two-fluorophore labels configured such thatone fluorophore acts as a donor and the other as an acceptor for Försterresonance energy transfer) may increase the space of potential labelsand/or the range of possible optical properties thereof. However, suchlabels may still be limited (e.g., with respect to the magnitude of theeffective Stokes shift of the label or other properties) by theavailability of resonators having the desired optical properties thatare also able to engage in energy transfer between each other (e.g., dueto having sufficiently overlapping emission and excitation spectra).

In order to provide more freedom to specify such optical properties of alabel and/or resonator network, the resonator network could include oneor more mediating resonators configured to allow energy to betransferred, from an input resonator, to an output resonator via the oneor more mediating resonators. In such resonator networks, the inputresonator and output resonator may be selected (e.g., according toexcitation spectrum/Wavelength, emission spectrum/wavelength,brightness, compatibility with environmental conditions, tendency tophotobleach) without the requirement that the output resonator be ableto receive energy directly (e.g., via resonance energy transfer) fromthe input resonator. The one or more mediating resonators can then beselected and located within the resonator network, relative to the inputand output resonators, such that energy received into the network as aresult of the resonator network being illuminated may be transmitted tothe output resonator via the mediating resonator(s).

FIG. 6A illustrates a schematic of an example resonator network 600 a asdescribed herein. The example resonator network 600 a includes an inputresonator (“IN”), a mediating resonator (“M1”), and an output resonator(“OUT”). The input resonator can be excited by receiving illuminationfrom the environment of the resonator network 600 a (e.g., illuminationat an excitation wavelength of the input resonator). The inputresonator, output resonator, and mediating resonator are arranged suchthat the mediating resonator can receive energy from the input resonatorand the output resonator can receive energy from the mediatingresonator. The mediating resonator may be selected (e.g., from a set ofcommercially available fluorophores) such that it is able to receiveenergy from the input resonator and provide energy to the outputresonator. This could include selecting the mediating resonator suchthat an emission spectrum of the input resonator overlaps with anexcitation spectrum of the mediating resonator and/or such that anemission spectrum of the mediating resonator overlaps with an excitationspectrum of the output resonator.

In order to permit a greater difference between the excitationspectrum/wavelength of the input resonator and the emissionspectrum/wavelength of the output resonator of such a resonator network,the resonator network could include additional mediating resonators(e.g., disposed as a resonator wire within the label) FIG. 6Billustrates a schematic of an example resonator network 600 b asdescribed herein. The example resonator network 600 b includes an inputresonator (“IN”), n mediating resonators (“M1” through “M4” and “Mn”),and an output resonator (“OUT”). The input resonator can be excited byreceiving illumination from the environment of the resonator network 600b (e.g., illumination at an excitation wavelength of the inputresonator). The mediating resonators are arranged as a resonator wire orarbitrary length between the input resonator and the output resonator.That is, the n mediating resonators are arranged such that eachresonator in the wire can receive energy from one neighboring resonatorand transmit energy to another neighboring resonator. The number andidentity of the resonators within such a resonator wire could bespecified in order to adjust a difference between an excitation spectrumof the input resonator and an emission spectrum of the output resonator,e.g., to adjust a difference between an excitation wavelength of theinput resonator and an emission wavelength of the output resonator. Insuch examples, each mediating resonator disposed between the input andoutput resonators could have an emission wavelength that is intermediatebetween an excitation wavelength of the input and an emission wavelengthof the output resonator, e.g., such that transfer of energy to and/orfrom each mediating resonator permits a controlled reduction in thewavelength and/or magnitude of an exciton (or other quantum) of energyfrom the input resonator to the output resonator.

Further, it may be beneficial to increase or otherwise specify thebrightness of labels and/or resonator networks as described herein inorder to facilitate the detection or identification of such resonatornetworks. For example, different analytes of interest in a sample may bepresent in the sample at different concentrations or amounts. In suchexamples, the number or concentration of proteins, receptors, smallmolecules, segments of RNA, segments of DNA, or other analytes ofinterest present a sample (e.g., a sample containing cells that may bedetected, identified, and/or sorted by a flow cytometry apparatus) maydiffer by a large amount (e.g., by multiple orders of magnitude). Insuch examples, applying a contrast agent that includes two labels,having approximately the same brightness, to the sample may result inthe brightness of a first one of the labels, configured to bind to themore prevalent analyte, being much greater than the brightness of asecond label, configured to bind to the less prevalent analyte, that isthus present in the sample at a lower concentration. The greaterbrightness, in the sample, of the first label may prevent or degrade thedetection of the second label in the sample. In such an example, it canbe beneficial to configure the second label to have a greater brightnessthan the first label. However, control over the brightness of such alabel may be constrained by a limited library of commercially orotherwise available resonators (e.g., fluorophores).

Additionally, it can be generally beneficial to increase the brightnessof resonator networks as described herein in order to facilitate thedetection of rare analytes, to reduce an intensity of illuminationnecessary for such detection (e.g., to reduce photobleaching of thelabels and/or to prevent damage to the sample due to such illumination),or to reduce an intensity of illumination necessary for some otherapplication of the resonator networks (e.g., performance of opticallogic functions, generation of samples of a random variable).

In order to increase or otherwise specify the brightness of suchresonator networks (e.g., relative to other labels present in a contrastagent), a resonator networks could be configured to have multiple inputresonators, output resonators, and/or resonator networks as describedherein. The ability to control the brightness of such a resonatornetworks, or of multiple different resonator networks (e.g., respectivedifferent resonator networks of two or more labels present in a contrastagent used for flow cytometry, molecular imaging, or some otherapplication) could facilitate panel selection for flow cytometry (e.g.,by permitting the specification of greater brightness of labelscorresponding to lower-abundance analytes in a sample relative to labelscorresponding to more prevalent analytes) or other applications.

In order to control the brightness of a resonator network, DNAself-assembly or other techniques could be used to provide a resonatornetwork having many instances of a single resonator, or of a number ofresonators, such that the overall brightness of the resonator networksis increased by an amount related to the number of instances of theresonator. This could include providing many copies of a resonatornetwork as described herein (e.g., 100, 300 a-f, 600 a-b) in order toincrease the effective brightness of such a label, to reduce the numberof photons detected therefrom and/or time (e.g., number of pulses ofillumination) necessary to identify such labels, or to provide someother benefit. Such multiple resonators and/or multiple resonatornetworks could be located sufficiently far apart, within a label, suchthat substantially no energy transfer (e.g., resonance energy transfer)occurs between the resonators and/or resonator networks. Additionally oralternatively, the resonators and/or resonator networks could engage inenergy transfer (e.g., to provide an increase in the brightness of theresonator networks via energy pooling or some other mechanism, or toprovide some other benefit).

Additionally or alternatively, an absolute or relative number of inputfluorophores and/or output fluorophores of a label and/or of a resonatornetwork of a label could be specified to control the overall brightnessof the resonator network. This could include specifying a resonatornetwork such that one output resonator may receive energy (e.g.,excitons) from a plurality of input resonators and/or such that a singleinput resonator may provide energy (e.g., excitons) to a plurality ofoutput resonators. For example, FIG. 6C illustrates a schematic of anexample resonator network 600 c that includes six input resonators(“IN1” through “IN6”) and an output resonator (“OUT”). As indicated bythe representative arrows, energy transfers may occur from each of theinput resonators directly to the output resonator. Such a resonatornetwork could provide increased brightness by increasing the absorptioncross-section of the resonator network, by providing additional sitesthat may be excited by illumination, or via some other mechanism orprocess.

In another example, FIG. 6D illustrates a schematic of an exampleresonator network 600 d that includes six output resonators (“OUT1”through “OUT6”) and an input resonator (“IN”). As indicated by therepresentative arrows, energy transfers may occur to each of the outputresonators directly from the input resonator. Such a resonator networkcould provide increased brightness in examples where energy transferfrom the input resonator to the output resonator is improbable, where atime to emission of light by the output resonators (e.g., a fluorescencelifetime) is long, or via some other mechanism or process.

Note that resonator networks as described herein may include both inputresonators that can provide energy to multiple output resonators andoutput resonators that can receive energy from multiple inputresonators. For example, FIG. 6E illustrates a schematic of an exampleresonator network 600 e that includes ten input resonators (“IN1”through “IN10”) and two output resonators (“OUT1” and “OUT2”). Asindicated by the representative arrows, energy transfers may occur from“IN1” and “IN2” directly to either of the output resonators. Energytransfers may also occur directly from “N3” through “IN6” to “OUT1” andfrom “IN7” through “IN10” to “OUT2.”

In some examples, a resonator network could include one or moremediating resonators (e.g., to increase a difference between anexcitation wavelength of an input resonator and an emission wavelengthof an output resonator, to adjust a temporal decay profile of theresonator network) to transfer energy from multiple input resonators toan output resonator and/or to transfer energy from an input resonator tomultiple output resonators. FIG. 6F illustrates a schematic of anexample resonator network 600 f as described herein. The exampleresonator network 600 f includes five input resonators (“IN1” through“IN5”), two mediating resonators (“M1” and “M2”), and an outputresonator (“OUT”). The input resonators can be excited by receivingillumination from the environment of the resonator network 600 f (e.g.,illumination at an excitation wavelength of the input resonators).

The two mediating resonators are arranged as a resonator wire betweenthe input resonators and the output resonator. That is, the twomediating resonators are arranged such that the first mediatingresonator can receive energy from each of the input resonators, thesecond mediating resonator can receive energy from the first mediatingresonator, and the output resonator can receive energy from the secondmediating resonator. The number of resonators within such a resonatorwire could be specified in order to adjust a temporal decay profile ofthe resonator network 600 f (e.g., to adjust a delay or width of a peakin the decay profile, to increase an average decay of the decay profile,or to adjust some other property of the temporal decay profile), toincrease a difference between an excitation wavelength of the inputresonators and an emission wavelength of the output resonator, or toprovide some other benefit.

The resonator network of a resonator network as described herein couldrepresent different topologies, e.g., a branched topology. Such abranched topology could include multiple different resonator wires whoseends are connected to input resonators, output resonators, mediatingresonators (e.g., an end resonator of one or more other resonatorwires), or connected in some other way to provide a resonator networkexhibiting a desired temporal decay profile.

In some examples, a label and/or resonator network could include aplurality of input resonators, mediating resonators, and/or outputresonators that are in some way interconnected to provide some or all ofthe benefits described herein. For example, FIG. 6G illustrates aschematic of an example resonator network 600 g that includes a field ofoutput resonators (“OUT”) and input resonators (“IN”). As indicated bythe representative arrows, energy transfers may occur to each of theoutput resonators directly from a number of input resonators and fromeach input resonator to one or more output resonators.

The brightness of such a resonator network, or of other resonatornetworks described herein (e.g., 600 c, 600 d, 600 e, 600 f) could beadjusted by controlling a ratio between a number of input resonators inthe network and a number of output resonators in the network. Forexample, for certain input resonators, output resonators, andenvironmental conditions, a brightness of a resonator network could beincreased by increasing a ratio between the number of input resonatorsand the number of output resonators (i.e., increasing the number ofinput resonators relative to the number of output resonators). Thus, therelative brightness of two labels comprising a contrast agent (e.g., acontrast agent used to stain a sample of cells for flow cytometry) couldbe adjusted by adjusting the ratios between input and output resonatorsof the two labels (e.g., such that a first ratio between inputresonators and output resonators of the first label differs from asecond ratio between input resonators and output resonators of thesecond label by a specified amount).

The brightness of a label and/or resonator network could also beincreased by providing a network of input resonators wherein energyreceived (e.g., from environmental illumination) by an input resonatorof the network is transferred to an output resonator of the network viaone or more additional input resonators. A field of such inputresonators could act to increase the absorption cross-section of theresonator network by effectively absorbing a significant fraction ofphotons that intersect with a planar shape and/or three-dimensionalvolume defined by the field of input resonators. Further, the inputresonators could exhibit bidirectional energy transfer (e.g., pairs ofneighboring input resonators could be capable of transferring energybetween themselves in either direction), allowing the field ofresonators to exhibit pooling of absorbed energy. Such pooling canincrease the probability that photons intersecting the field areabsorbed and/or increase the probability that energy absorbed by thefield are successfully transferred, via the overall resonator network,to an output resonator. Such a resonator network could include manyinput resonators per output resonator, e.g., more than four inputresonators per output resonator, or more than thirty input resonatorsper output resonator. The input resonators of such a field of inputresonators could all be the same type of input resonator (e.g., the sametype of fluorophore, having excitation and emission spectra that overlapsuch that different instances of the fluorophore can transmit energybetween each other) or different types of resonators (e.g., to permitabsorption of photons at multiple different excitation wavelengths or toprovide some other benefit).

For example, FIG. 6H illustrates a schematic of an example resonatornetwork 600 h that includes an output resonator (“OUT”) and a field ofinput resonators (“IN”). As indicated by the representative arrows,energy transfers may occur, bidirectionally, between neighboring inputresonators. Additionally, energy transfer may occur to the outputresonator directly from a number of neighboring input resonators.Accordingly, the output resonator may receive energy indirectly fromnon-neighboring input resonators via energy transmission throughintermediary input resonators.

VI. EXAMPLE LOGICAL RESONATOR NETWORKS

Resonator networks as described here (e.g., that are part of labels,that are used to generate random number generators) may be configured toexhibit behaviors that are optically modulatable or otherwisecontrollable. In some examples, the network behavior could be opticallycontrollable, allowing the network to perform logical operations or toprovide some other benefits. Such optical control could be provided forby one or more resonators of the network having an optically-induceable“dark state,” wherein the resonator is unable, or less able, to transmitand/or receive energy (e.g., excitons) when in the dark state.Additionally or alternatively, the behavior of a resonator network couldbe related to a property of the environment of the network (e.g., to apH level, to the binding of an analyte of interest to the network),permitting the resonator network to be used to optically detect theproperty of the environment of the network. In some examples, a singleresonator network could include both sensor behaviors andoptically-controllable behaviors, allowing a single resonator network tobe optically controlled to detect multiple different analytes or otherenvironmental variables (e.g., by operating optical logic elements ofthe network to “address” a particular sensed variable of interest).

Optical control of resonator network behavior can be provided via avariety of methods. In some examples, the state of individual resonatorsmay be optically adjusted. This may be performed irreversibly. e.g., byphotobleaching one or more resonators by illuminating the resonatorswith illumination at an excitation wavelength of the resonator(s) at anintensity above a threshold level. Alternatively, the state ofindividual resonators may be reversibly adjusted, e.g., by opticallyinducing the resonator(s) to enter a “dark state.”

A “dark state” is a state wherein a resonator (e.g., a fluorophore, aquantum dot, or some other optically active molecule or atom asdescribed herein) become incapable, or become less capable, oftransmitting and/or receiving energy (e.g., photons, excitons) to and/orfrom the environment of the resonator (e.g., from other resonators of aresonator network). The resonator may be optically placed into the darkstate by illumination by light at a particular wavelength. Suchillumination may cause the resonator to enter the dark state by, e.g.,causing an electron to transition into another energy state thatprevents the resonator from absorbing additional energy, causing theresonator to gain/lose charge (e.g., to receive and/or donate anelectron from/to the environment), or by causing the resonator toundergo some other process. Accordingly, a resonator network thatincludes one or more such resonators (i.e., resonators that may beoptically controlled to enter a dark state) may have a temporal decayprofile, a probability of photon re-emission following illumination, orsome other property that is optically controllable by providingillumination sufficient to cause the resonator(s) to enter the darkstate.

Such dark state resonators may be provided as part of a resonatornetwork in order to allow for optical control of the flow of energy(e.g., excitons) through the network. Such a resonator network could beconfigured such that the dark state resonator, when in the dark state,acts to facilitate energy flow through the resonator network (e.g., fromone portion of the network to another, and/or from an input of thenetwork to an output of the network). Additionally or alternatively, aresonator network could be configured such that the dark stateresonator, when in the dark state, acts to inhibit energy flow throughthe resonator network (e.g., from one portion of the network to another,and/or from an input of the network to an output of the network). Suchoptically-controllable inhibition and/or excitation can be used toprovide logic gates, energy flow control within a resonator network, ora variety of other behaviors and/or applications.

A resonator that exhibits such an optically-inducible dark state may beapplied in a variety of ways within a resonator network in order to,when in the dark state, inhibit energy flow through the resonatornetwork. For example, such an inhibiting resonator may be provided aspart of a path for energy flow within the resonator network.Accordingly, when the inhibiting resonator is in the dark state (e.g.,due to illuminating the resonator network with light at an appropriatewavelength), energy flow along the path will be fully or partiallyprevented, thus fully or partially inhibiting energy flow along thepath.

This is illustrated by way of example in FIGS. 7A and 7B, whichillustrate an example resonator network 700 a at respective differentpoints in time. The resonator network 700 a includes a readout resonator(“CLK”), an input resonator (“IN”), and an output resonator (“OUT”).When the input resonator is not in the dark state (illustrated in FIG.7A), energy (e.g., excitons) may be transmitted from the readoutresonator (e.g., in response to the readout resonator being illuminatedby light 710 a at an excitation wavelength of the readout resonator) tothe input resonator, and from the input resonator to the outputresonator. Thus, when the input resonator is not in the dark state,illumination 710 a absorbed by the resonator network (by the readoutresonator) may be transmitted through the resonator network 700 a to theoutput resonator, and then emitted as a photon 720 a from the outputresonator.

Conversely, when the input resonator is in the dark state (illustrated,in FIG. 7B, by the “IN” resonator being drawn in dashed lines), energy(e.g., excitons) is unable to be transmitted from the readout resonatorto the input resonator, and from the input resonator to the outputresonator. Thus, when the input resonator is in the dark state,illumination 710 a absorbed by the resonator network (by the readoutresonator) is not transmitted through the resonator network 700 a to theoutput resonator, which thus does not responsively emit a photon.

Additionally or alternatively, a resonator that exhibits such anoptically-inducible dark state may be applied in a variety of wayswithin a resonator network in order to, when in the dark state,facilitate energy flow through the resonator network. For example, sucha facilitating resonator may be provided as part of an alternative,dissipative and/or non-radiative path for energy flow within theresonator network. Such a facilitating resonator, which not in the darkstate, could act to sink or otherwise preferentially receive energy(e.g., excitons), preventing the energy from traveling to an outputresonator or other portion of the resonator network. Accordingly, whenthe facilitating resonator is in the dark state (e.g., due toilluminating the resonator network with light at an appropriatewavelength), energy will not flow to the facilitating resonator and thusmay flow along a different path through the network (e.g., to an outputresonator).

This is illustrated by way of example in FIGS. 7C and 7D, whichillustrate an example resonator network 700 b at respective differentpoints in time. The resonator network 700 b includes a readout resonator(“CLK”), an input resonator (“IN”), a mediating resonator (“M”), and anoutput resonator (“OUT”). When the input resonator is not in the darkstate (illustrated in FIG. 7C), energy (e.g., excitons) may betransmitted from the readout resonator (e.g., in response to the readoutresonator being illuminated by light 710 b at an excitation wavelengthof the readout resonator) to the mediating resonator, and from themediating resonator to either of the input resonator or the outputresonator. If transmitted to the input resonator, the energy is likelyto be dissipated (e.g., lost from the network as heat, or emitted as aphoton at an emission wavelength of the input resonator), while theenergy, if transmitted to the output resonator, is likely to be emittedas a photon, at an emission wavelength of the output resonator, from theoutput resonator.

The relative probability of the different energy transfers between theresonators are indicated in FIGS. 7C and 7D by the relative line weightof their representative arrows. Thus, for the example network 700 b,when the input resonator is not in the dark state, it is more likelythat the mediating resonator transfers energy to the input resonatorthan to the output resonator. Thus, when the input resonator is not inthe dark state, illumination 710 b absorbed by the resonator network (bythe readout resonator) is more likely to be absorbed, and thendissipated by, the input resonator than it is to be received by theoutput resonator and transmitted from the network 700 b as a photon.

Conversely, when the input resonator is in the dark state (illustrated,in FIG. 7D, by the “IN” resonator being drawn in dashed lines), energy(e.g., excitons) is unable to be transmitted from the mediatingresonator to the input resonator, and thus is transmitted to the outputresonator. Thus, when the input resonator is in the dark state,illumination 710 b absorbed by the resonator network (by the readoutresonator) may be transmitted through the resonator network 700 b to theoutput resonator, and then emitted as a photon 720 b from the outputresonator.

Such behavior may be employed to implement logic gates or othercomputational or gating functions in a resonator network as describedherein. For example, the resonator network 700 a illustrated in FIGS. 7Aand 7B could be employed as a NOT gate, with “evaluation” of the gatetriggered by excitation of the readout resonator. Detection of the gateoutput may be achieved by detecting whether the output resonator emitteda photon in response to the “evaluation.” The gate input is applied byproviding (or not providing) illumination at an input wavelength suchthat the input resonator enters the dark state. Accordingly, a “high”input (illumination sufficient to cause the input to enter the darkstate) would result in a “low” output (the network not emitting a photonfrom the output resonator in response to excitation of the readoutresonator) Conversely, a “low” input will result in a “high” output,providing the behavior of a NOT gate.

Resonator structures may be designed to provide arbitrary logic gatefunctions or other computational or gating functionality. This caninclude providing multiple “input” resonators, which may be caused toenter a dark state by providing illumination to the input resonators atan appropriate wavelength. These additional input resonators may differwith respect to the wavelength of light necessary to induce the darkstate. These additional resonators may also differ with respect towhether they facilitate the flow of energy through the network orinhibit the flow of energy through the network. Accordingly, lightprovided (or not provided) at these different wavelengths may representrespective different logical inputs to the resonator network. Thewavelengths may differ by more than a specified amount (e.g., by morethan 10 nanometers, or by more than 50 nanometers) in order to permitreliable and independent signaling along the respective differentlogical inputs.

An example of such a resonator network, configured as a logical ANDgate, is shown in FIG. 8A. The resonator network 800 a includes areadout resonator (“CLK”), two mediating resonators (“M1” and “M2”), twoinput resonators (“IN1” and IN2”), and an output resonator (“OUT”). Therelative probability of the different energy transfers between theresonators are indicated in FIG. 8A by the relative line weight of theirrepresentative arrows. Thus, in order for energy to be transmitted fromthe readout resonator to the output resonator with high probability,both of the input resonators must be in their dark states (e.g., inresponse to being provided with illumination at their respective inputwavelengths).

Another example of such a resonator network, configured as a logical ORgate, is shown in FIG. 8B. The resonator network 800 b includes areadout resonator (“CLK”), two mediating resonators (“M1” and “M2”), twoinput resonators (“IN1” and IN2”), and an output resonator (“OUT”). Therelative probability of the different energy transfers between theresonators are indicated in FIG. 8B by the relative line weight of theirrepresentative arrows. Thus, in order for energy to be transmitted fromthe readout resonator to the output resonator with high probability, atleast one of the input resonators must be in its dark state (e.g., inresponse to being provided with illumination at one or both of theirrespective input wavelengths).

Another example of such a resonator network, configured as a logicalNAND gate, is shown in FIG. 8C. The resonator network 800 c includes areadout resonator (“CLK”), two input resonators (“IN1” and IN2”), and anoutput resonator (“OUT”). The relative probability of the differentenergy transfers between the resonators are indicated in FIG. 8C by therelative line weight of their representative arrows. Thus, in order forenergy to be transmitted from the readout resonator to the outputresonator with high probability, no more than one of the inputresonators may be in its dark state (e.g., in response to being providedwith illumination at one or the other, or neither, of their respectiveinput wavelengths).

Another example of such a resonator network, configured as a logical NORgate, is shown in FIG. 8D. The resonator network 800 d includes areadout resonator (“CLK”), two input resonators (“IN1” and IN2”), and anoutput resonator (“OUT”). The relative probability of the differentenergy transfers between the resonators are indicated in FIG. 8D by therelative line weight of their representative arrows. Thus, in order forenergy to be transmitted from the readout resonator to the outputresonator with high probability, neither of the input resonators may bein their dark states (e.g., in response to being provided withillumination at neither of their respective input wavelengths).

Multiple input resonators that enter their dark states in response toreceiving illumination at the same wavelength may be provided in asingle resonator network in order to achieve a specified logicalfunction or behavior. An example of such a resonator network, configuredas a logical XOR gate, is shown in FIG. 8E. The resonator network 800 eincludes a readout resonator (“CLK”), two mediating resonators (“M1” and“M2”), four input resonators (“IN1a,” “IN1b,” “IN2a,” and IN2b”), and anoutput resonator (“OUT”). The relative probability of the differentenergy transfers between the resonators are indicated in FIG. 8E by therelative line weight of their representative arrows. Thus, in order forenergy to be transmitted from the readout resonator to the outputresonator with high probability, one and only one of the inputresonators must be in their dark state (e.g., in response to beingprovided with illumination at one or the other, exclusively, of theirrespective input wavelengths).

Another example of such a resonator network, configured as a logicalXNOR gate, is shown in FIG. 8F. The resonator network 800 f includes areadout resonator (“CLK”), two mediating resonators (“M1” and “M2”),four input resonators (“IN1a,” “IN1b,” “IN2a,” and IN2b”), and an outputresonator (“OUT”). The relative probability of the different energytransfers between the resonators are indicated in FIG. 8F by therelative line weight of their representative arrows. Thus, in order forenergy to be transmitted from the readout resonator to the outputresonator with high probability, either both or neither of the inputresonators must be in their dark state (e.g., in response to beingprovided with illumination at both of their respective input wavelengthsor at neither of their respective input wavelengths).

A resonator network may include input resonators as described herein(e.g., dark-state-exhibiting resonators whose dark state may beoptically induced and/or otherwise optically controlled) to control theflow of energy through the resonator network (e.g., between differentportions of the resonator network). Such inputs may be controlled inorder to selectively activate or deactivate portions of the resonatornetwork. This is illustrated by way of example in FIG. 9A, which shows aresonator network 900 a that includes a readout resonator (“CLK”), threeinput resonators (“IN1,” “IN2,” and IN3”), and three output resonators(“OUT1,” “OUT2,” and “OUT3”). The relative probability of the differentenergy transfers between the resonators are indicated in FIG. 9A by therelative line weight of their representative arrows. Thus, in order forenergy to be transmitted from the readout resonator to a particular oneof the output resonators, the corresponding input resonator must not bein its dark state. Accordingly, the output resonator(s) that may emitphotons in response to excitation of the readout resonator may byselected by providing (or not providing) illumination at the respectiveinput wavelengths of the input resonators. For example, to select the“OUT1” output resonator, light could be provided at thedark-state-inducing wavelengths for the second (“IN2”) and third (“IN3”)input resonators.

Resonator networks that are optically-controllable (e.g., by opticallyinducing a dark state in one or more resonators of the networks) may beapplied to provide a variety of benefits. For example, resonatornetwork-containing labels as described herein may include such darkstate resonators in order to provide further multiplexing for labeldetection and identification. This could include the label exhibiting afirst temporal decay profile or other time-dependent probability densityfunction with respect to the relative timing of emission of photons inresponse to illumination when an input resonator of the label is in adark state. The label could then exhibit a second temporal decay profileor other time-dependent probability density function when the inputresonator is not in the dark state. Accordingly, the label could beoptically interrogated during the first and second periods of time, withthe input resonator being not in the dark state during the first periodof time and being in the dark state during the second period of time(e.g., due to illumination at an excitation wavelength of the inputresonator) The detected relative timing of emission of light from thelabel, in response to illumination, during the first and second timeperiods could be used together to identify the label.

In another example, resonator networks as described herein may includesuch dark state resonators in order to provide a controllabletime-dependent probability density function with respect to the timingof emission of photons from the resonator network(s) in response toillumination. The detected relative timing could be used to generatesamples of a random variable, with the probability distribution of therandom variable being related to the time-dependent probability densityfunction exhibited by the resonator network(s). One or more inputresonators of such a resonator network being in a dark state couldmodify the time-dependent probability density function exhibited by theresonator network(s). Accordingly, the probability distribution of therandom variable samples generated therefrom could be controlled bycontrolling whether such input resonator(s) are in the dark state.

In some examples, this could include applying dark state inputresonators within a resonator network to control whether sections of theresonator network are available to transfer energy from a readoutresonator of the network to an output resonator of the network. Eachsuch configuration of the network, including only the portions of thenetwork “enabled” by the dark state of the input resonator(s), couldcorrespond to a respective different time-dependent probability densityfunction and thus be used to generate samples of a respective differentrandom variable.

This is illustrated by way of example in FIG. 9B, which shows aresonator network 900 b that includes a readout resonator (“CLK”), threeinput resonators (“IN1,” “IN2,” and IN3”), twelve mediating resonators(“M1” through “M12”), and an output resonator (“OUT”). The relativeprobability of the different energy transfers between the resonators areindicated in FIG. 9B by the relative line weight of their representativearrows. Thus, in order for energy to be transmitted from the readoutresonator to the output resonator, at least one of the input resonatorsmust not be in its dark state. The overall time-dependent probabilitydensity function exhibited by the resonator network 900 b, with respectto the timing of emission of photons from the output resonator inresponse to excitation of the readout resonator, is related to whethereach of the input resonators is or is not in its dark state. So, forexample, if the “IN2” and “IN3” input resonators are in their darkstate, and “IN1” is not in its dark state, the resonator network 900 bwill exhibit a time-dependent probability density function related tothe resonator wire comprised of “IN1,” “M1,” M2,” “M3,” and “M4.” Inanother example, if the “IN3” input resonator is in its dark state, andthe “IN1” and “IN2” resonators are not in their dark states, theresonator network 900 b will exhibit a time-dependent probabilitydensity function related to a combination of the time-dependentprobability density function of the resonator wire comprised of N1,”“M1,” M2,” “M3,” and “M4” and an additional time-dependent probabilitydensity function related to the resonator wire comprised of “IN2,” M5,”and “M6.”

In yet another example, resonator networks as described herein mayinclude sensors for detecting properties of the environment of theresonator networks, e.g., a pH of a solution to which the resonatornetwork is exposed, or the presence or amount of an analyte bound to areceptor of the resonator network. Such a resonator network couldinclude a variety of sensor elements or other components (e.g., theresonators of the network itself) that are able to transduce a propertyof the environment of the network into an optically-detectable change inthe resonator network (e.g., a change in an overall intensity orprobability of light emission in response to illumination, a change in atemporal decay function and/or a time-dependent probability densityfunction of light emission from the network in response toillumination). For example, one or more resonators of the resonatornetwork may have an optical property (e.g., a property of beingquenched, or of entering a dark state) that is related to a pH or otherproperty of a solution to which the resonator is exposed, to whether theresonator has bound to an analyte of interest, or to some other propertyof interest in the environment of the resonator network.

In another example, such a sensor could comprise a receptor (e.g., anantibody, an aptamer, one or more proteins, a DNA or RNA strand) thatpreferentially binds to an analyte of interest (e.g., a protein, ahormone, a cell, a cell surface receptor or other cell surface element,a complementary DNA or RNA strand, a small molecule, a metal ion). Thestate of binding of such a receptor to the analyte of interest couldthen be related to one or more detectable optical properties of theresonator network in a variety of ways. For example, binding of theanalyte to the receptor could result in a change of the relativelocation of one or more resonators within the resonator network, thuschanging an optically detectable property of the resonator network(e.g., a overall intensity or probability of light emission in responseto illumination, a change in a temporal decay function and/or atime-dependent probability density function of light emission from thenetwork in response to illumination). Such a change could be due to achange in conformation of the receptor, to a change in conformation ofone or more elements of a backbone of the resonator network, or to achange in location of a resonator or backbone element coupled to thereceptor. Additionally or alternatively, the receptor could be coupledto and/or part of a resonator of the network (e.g., part of a proteinthat includes a fluorescent moiety) such that the receptor not beingbound to an instance of the analyte causes the resonator to be quenchedor otherwise optically disabled. Alternatively, the receptor being boundto an instance of the analyte could cause the resonator to be quenchedor otherwise optically disabled.

This is illustrated by way of example in FIGS. 10A and 10B, whichillustrate an example resonator network 1000. The resonator network 1000includes a readout resonator (“CLK”), a receptor 730 that preferentiallybinds to an analyte of interest 735, a mediating resonator (“IN”) thatis quenched when an instance of the analyte 735 is bound to the receptor730, and an output resonator (“OUT”). Thus, when the receptor 730 is notbound to an instance of the analyte, the resonator network 1000 can emitlight 720 a in response to receiving light 710 a at an excitationwavelength of the readout resonator (illustrated in FIG. 10A).Conversely, when the receptor 730 is bound to an instance of the analyte735, the resonator network 1000 is unable to emit light in response toreceiving light 710 a at an excitation wavelength of the readoutresonator, since the mediating resonator has been quenched and is thusunavailable to transmit received energy from the readout resonator tothe output resonator (illustrated in FIG. 10B).

A resonator network that is configured, as described above, foroptically sensing one or more properties of the environment of theresonator network may include one or more dark state exhibiting inputresonators. Such input resonators could permit multiplexing of theresonator network in order to use the network to detect multipledifferent environmental properties. For example, a resonator couldinclude multiple different receptors that selectively interact withrespective different analytes and that, when bound to an instance of arespective analyte, quench a respective resonator of the network orotherwise induce a change in an optical property of a respective portionof the resonator network. One or more input resonators could be providedin such a resonator network, to permit optically-controlled multiplexingof analyte detection using the resonator network. This could includeusing the input resonators to implement logic gates or other means foraddressing the sensors such that the resonator network response to areadout resonator being excited (e.g., an intensity or a timing ofemission of light from an output resonator of the resonator network) isrelated to whether an optically-selected one of the receptors is boundto an instance of a corresponding analyte. Such optically-controlledmultiplexing could also permit sub-wavelength imaging and/or analyteassays, by enabling the optical control and/or selection of differentportions of a resonator network that are separated from each other by adistance that is less than an imaging wavelength.

VII. EXAMPLE METHODS

FIG. 11 is a flowchart of a method 1100 for interrogating a sample todetect and identify one or more labels, as described herein, that may becontained within the sample. For purposes of illustration, the labelidentified in method 1100 includes: (i) an input resonator; (ii) anoutput resonator that is characterized by an emission wavelength; and(iii) a network of one or more mediating resonators. The relativelocations of the input resonator, the output resonator, and the one ormore mediating resonators within the label are such that energy can betransmitted from the input resonator to the output resonator via thenetwork of one or more mediating resonators in response to the inputresonator being excited by illumination (e.g., by a pulse of laser lightat an excitation wavelength of the input resonator).

The method 1100 includes illuminating a sample that contains the label(1110). This could include illuminating the sample with one or morepulses of illumination. Such pulses of illumination could be ultrashortpulses, having pulse widths between attoseconds and nanoseconds. Thepulses of illumination could have different spectra and/or includedifferent wavelengths of light. For example, a first pulse ofillumination could include light at an excitation wavelength of theinput resonator of the label and a second pulse of illumination couldinclude light at an excitation wavelength of an input resonator of adifferent label. In another example, a first pulse of illumination couldinclude light at an excitation wavelength of the input resonator of thelabel and a second pulse of illumination could include light at anexcitation wavelength of a further input resonator of the label.

The method 1100 also includes detecting a timing, relative to theillumination of the sample, of emission of a plurality of photons fromthe sample within a range of detection wavelengths (1120). The range ofdetection wavelengths includes the emission wavelength of the outputresonator of the label. Detecting the timing of emission of a pluralityof photons from the sample could include detecting the timing ofreception of individual photons, e.g., using a single photon avalanchediode, a photomultiplier tube, or some other detector element(s).Additionally or alternatively, detecting the timing of emission of aplurality of photons from the sample could include detecting a timing ofa peak or other feature of the variation over time of the intensity,rate, or other property of the photons emitted from the sample.

The method 1100 further includes determining, based on the detectedtiming of emission of the plurality of photons, an identity of the label(1130). Determining the identity of the label includes selecting theidentity of the label from a set of known labels. Determining theidentity of the label could include comparing the detected timing ofemission of the plurality of photons to a set of temporal decay profilesthat correspond to the known labels. For example, the detected timing ofemission of the plurality of photons could be used to determine aprobability density function for the timing of emission of photons fromthe sample in response to illumination of the sample. Such a determinedprobability density function could then be compared to each of the knowntemporal decay profiles. Such a comparison could include determining ameasure of statistical divergence between the probability densityfunction and the known temporal decay profiles, e.g., a Kullback-Leiblerdivergence, a Jensen-Shannon divergence, a Bregman divergence, or aFisher information metric.

The method 1100 could include additional or alternative steps asdescribed elsewhere herein. For example, the method 1100 could includeidentifying a cell or other contents of the sample based on thedetermined identity of one or more labels in the sample. The method 1100could include sorting cells or other particulates in the sample, basedon the determined identity of the label (e.g., the sample could becontained within a flow channel of a flow cytometry apparatus, and cellsin the flow chamber could be sorted according to the determined identityof one or more labels in the flow channel). The method 1100 couldinclude emitting light at an excitation wavelength of a darkstate-exhibiting resonator of the resonator wavelength, such that thetemporal decay profile or other optically-detectable property of thelabel is adjusted, and identifying the label could include determiningthat the detected timing corresponds to the adjusted state of theoptically-detectable property. The example method 1100 illustrated inFIG. 11 is meant as an illustrative, non-limiting example. Additional oralternative elements of the method are anticipated, as will be obviousto one skilled in the art.

FIG. 12 is a flowchart of a method 1200 for interrogating a resonatornetwork as described herein to detect an analyte. For purposes ofillustrations, the resonator network of method 1200 includes: (i) afirst input resonator that has a dark state and that can enter the darkstate in response to receiving illumination at a first input excitationwavelength; (ii) a readout resonator that can receive energy fromillumination at a readout wavelength; (iii) a mediating resonator; (iv)an output resonator; (v) a sensor that includes a receptor thatpreferentially binds to the analyte, and (vi) a backbone. The firstinput resonator, the readout resonator, the sensor, and the outputresonator are coupled to the backbone. The backbone maintains relativelocations of the first input resonator, the readout resonator, themediating resonator, the sensor, and the output resonator such thatenergy can be transmitted from the readout resonator to the outputresonator via the mediating resonator and further such that aprobability of energy being transmitted from the readout resonator tothe output resonator, when the first input resonator is in the darkstate, is related to whether the receptor is bound to an instance of theanalyte.

The method 1200 includes illuminating the resonator network, during afirst period of time, with light at the first input wavelength (1210).This could include illuminating the sample with one or more pulses ofillumination. The duration and/or number of such pulses of suchillumination could be specified to ensure that the first input resonatoris likely to have entered the dark state. e.g., the provided light atthe first input wavelength could be provided for more than a thresholdduration of time.

The method 1200 includes illuminating the resonator network, during thefirst period of time, with light at the readout wavelength (1220). Thiscould include illuminating the sample with one or more pulses ofillumination. Such pulses of illumination could be ultrashort pulses,having pulse widths between attoseconds and nanoseconds. The pulses ofillumination could have different spectra and/or include differentwavelengths of light. For example, a first pulse of illumination couldinclude light at an excitation wavelength of the input resonator of thelabel and a second pulse of illumination could include light at anexcitation wavelength of an input resonator of a different label. Inanother example, a first pulse of illumination could include light at anexcitation wavelength of the input resonator of the label and a secondpulse of illumination could include light at an excitation wavelength ofa further input resonator of the label. The light at the readoutwavelength could be provided subsequent to providing the light at thefirst input wavelength.

The method 1200 also includes detecting, during the first period oftime, an intensity of light emitted from an output resonator of theresonator network (1230). This could include detecting a timing ofemission of such light relative to the timing of one or more pulses oflight provided at the readout wavelength. Detecting the intensity oflight emitted from the resonator network could include detecting atiming of emission of a plurality of photons from a population ofresonator networks, e.g., detecting the timing of reception ofindividual photons using a single photon avalanche diode, aphotomultiplier tube, or some other detector element(s). Additionally oralternatively, detecting the timing of emission of a plurality ofphotons from the sample could include detecting a timing of a peak orother feature of the variation over time of the intensity, rate, orother property of the photons emitted from the sample. Detecting theintensity of light emitted from the resonator network could includedetecting a total amount of light emitted from the output resonator,e.g., by integrating a signal related to the intensity of the detectedlight.

The method 1200 could include additional or alternative steps asdescribed elsewhere herein. The method 1200 could include determining,based on the detected intensity of the emitted light, a presence,amount, count, or other property of the analyte. In some examples, theresonator network could be configured to permit the detection ofmultiple analytes, e.g., by a process of optically multiplexing and/oraddressing multiple different sensors of the resonator network. Forexample, the resonator network could include a second sensor sensitiveto a second analyte and a second input resonator coupled together withthe remainder of the resonator network such that a probability of energybeing transmitted from the readout resonator to the output resonator,when the second input resonator is in the dark state and the first inputresonator is not in the dark state, is related to whether the secondreceptor is bound to an instance of the second analyte. In such anexample, the method 1200 could include, during a second period of time,illuminating the resonator network with light at an excitationwavelength of the second input resonator, illuminating the resonatornetwork with light at the readout wavelength; and detecting an intensityof light emitted from the resonator network during the second period oftime. The intensity detected during the second period of time could thenbe used to determine a concentration, a presence, a count, or some otherinformation about the second analyte. The example method 1200illustrated in FIG. 12 is meant as an illustrative, non-limitingexample. Additional or alternative elements of the method areanticipated, as will be obvious to one skilled in the art.

FIG. 13 is a flowchart of a method 1300 for using a plurality ofresonator networks, as described herein, to generate samples of a randomvariable. For purposes of illustration, the resonator network identifiedin method 1300 includes: (i) a first input resonator that has a darkstate and that can enter the dark state in response to receivingillumination at a first input wavelength; (ii) a readout resonator thatcan receive energy from illumination at a readout wavelength; (iii) twoor more mediating resonators; (iv) an output resonator; and (v) abackbone. The first input resonator, the readout resonator, the two ormore mediating resonators, and the output resonator are coupled to thebackbone. The backbone maintains relative locations of the first inputresonator, the readout resonator, the two or more mediating resonators,and the output resonator such that energy can be transmitted from thereadout resonator to the output resonator via the mediating resonatorand further such that the resonator network emits photons from theoutput resonator, in response to the readout resonator receivingillumination at the readout wavelength, according to a time-dependentprobability density function, and wherein a detectable property of thetime-dependent probability density function is related to whether thefirst input resonator is in the dark state.

The method 1300 includes illuminating the plurality of resonatornetworks, during a first period of time, with light at the first inputwavelength (1310). This could include illuminating the sample with oneor more pulses of illumination. The duration and/or number of suchpulses of such illumination could be specified to ensure that the firstinput resonator of each of the resonator networks and/or of a specifiedportion of the resonator networks is likely to have entered the darkstate, e.g., the provided light at the first input wavelength could beprovided for more than a threshold duration of time.

The method 1300 includes illuminating the plurality of resonatornetworks, during the first period of time, with light at the readoutwavelength (1320). This could include illuminating the sample with oneor more pulses of illumination. Such pulses of illumination could beultrashort pulses, having pulse widths between attoseconds andmicroseconds.

The method 1300 also includes detecting a timing, relative to theillumination of the resonator networks, of emission of a plurality ofphotons from the output resonators of the plurality of resonatornetworks (1330). Detecting the timing of emission of a plurality ofphotons from the resonator networks could include detecting the timingof reception of individual photons, e.g., using a single photonavalanche diode, a photomultiplier tube, or some other detectorelement(s). Additionally or alternatively, detecting the timing ofemission of a plurality of photons from the sample could includedetecting a timing of a peak or other feature of the variation over timeof the intensity, rate, or other property of the photons emitted fromthe sample.

The method 1300 could include additional or alternative steps asdescribed elsewhere herein. For example, the method 1300 could includegenerating a sample of a random variable based on the detected timing,e.g., by applying a function to the detected timing. The method 1300could include generating additional samples of the random variable byilluminating the resonator network and detecting a timing of emission ofphoton(s) responsively emitted from the resonator network. The resonatornetwork could include one or more additional input resonators, and themethod 1300 could include, during additional periods of time, generatingsamples of additional random variables by optically controlling theinput resonators of the resonator network such that the resonatornetwork exhibited time-dependent probability density functionscorresponding to the additional random variables. The samples of therandom variables could be generated by detecting a timing of emission oflight from the resonator network in response to illumination. Theexample method 1300 illustrated in FIG. 13 is meant as an illustrative,non-limiting example. Additional or alternative elements of the methodare anticipated, as will be obvious to one skilled in the art.

VIII. CONCLUSION

“Fluorescent taggants with temporally coded signatures” (Wang, S., Vyas,R., Dwyer, C, “Fluorescent taggants with temporally coded signatures.”Optics Express, Vol. 24, No. 14, 11 Jul. 2016) is incorporated herein byreference. All references cited herein are incorporated by reference. Inaddition, the invention is not intended to be limited to the disclosedembodiments of the invention. It should be understood that the foregoingdisclosure emphasizes certain specific embodiments of the invention andthat all modifications or alternatives equivalent thereto are within thespirit and scope of the invention as set forth in the appended claims

The particular arrangements shown in the Figures should not be viewed aslimiting. It should be understood that other embodiments may includemore or less of each element shown in a given Figure. Further, some ofthe illustrated elements may be combined or omitted. Yet further, anexemplary embodiment may include elements that are not illustrated inthe Figures.

Additionally, while various aspects and embodiments have been disclosedherein, other aspects and embodiments will be apparent to those skilledin the art. The various aspects and embodiments disclosed herein are forpurposes of illustration and are not intended to be limiting, with thetrue scope being indicated by the following claims. Other embodimentsmay be utilized, and other changes may be made, without departing fromthe spirit or scope of the subject matter presented herein. It will bereadily understood that the aspects of the present disclosure, asgenerally described herein, and illustrated in the figures, can bearranged, substituted, combined, separated, and designed in a widevariety of different configurations, all of which are contemplatedherein.

1-103. (canceled)
 104. A label comprising: a plurality of inputresonators, wherein input resonators of the plurality of inputresonators are disposed within the label proximate to each other suchthat the input resonators can transmit energy between each other; and abackbone, wherein the input resonators are coupled to the backbone, andwherein the backbone maintains relative locations of the inputresonators such that energy can be transmitted between the inputresonators.
 105. The label of claim 104, wherein the backbone comprisestwo strands of DNA that are at least partially complementary.
 106. Thelabel of claim 104, wherein all of the input resonators of the pluralityof input resonators comprise the same fluorophore.
 107. The label ofclaim 104, further comprising a plurality of output resonators, whereinoutput resonators of the plurality of output resonators are coupled tothe backbone, and wherein the backbone maintains relative locations ofthe input resonators and the output resonators such that energy can betransmitted to each of the output resonators from a respective inputresonator of the plurality of input resonators.
 108. The label of claim107, wherein the input resonators absorb light at an excitationwavelength, wherein the output resonators emits light at an emissionwavelength, and wherein the emission wavelength differs from theexcitation wavelength.
 109. The label of claim 104, further comprising:a receptor, wherein the receptor selectively interacts with an analyteof interest, and wherein the receptor is coupled to the backbone. 110.The label of claim 104, wherein the plurality of input resonatorscomprises more than four input resonators.
 111. The label of claim 104,wherein the plurality of input resonators comprises more than thirtyinput resonators.